Improved Production of Glucose Syrups

ABSTRACT

The present invention relates to a method of making glucose syrup from liquefied starch, and to compositions useful therein.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for production of glucose syrupcomprising high % DX from liquefied starch and to compositionscomprising enzymes for use therein.

BACKGROUND OF THE INVENTION

Starch usually consists of about 80% amylopectin and 20% amylose.Amylopectin is a branched polysaccharide in which linear chainsalpha-1,4 D-glucose residues are joined by alpha-1,6 glucosidiclinkages. Amylopectin is partially degraded by alpha-amylase, whichhydrolyzes the 1,4-alpha-glucosidic linkages to produce branched andlinear oligosaccharides.

Alpha-amylases are used commercially for a variety of purposes such asin the initial stages of starch processing (e.g., liquefaction).Prolonged degradation of amylopectin by alpha-amylase results in theformation of so-called alpha-limit dextrins that are not susceptible tofurther hydrolysis by the alpha-amylase. Alpha-amylases (1,4-α-D-glucanglucanohydrolase, EC 3.2.1.1) constitute a group of enzymes whichcatalyze hydrolysis of starch and other linear and branched1,4-glucosidic oligo- and polysaccharides.

Branched oligosaccharides can be hydrolyzed into linear oligosaccharidesby a debranching enzyme. The remaining branched oligosaccharides can bedepolymerized to D-glucose by glucoamylase, which hydrolyzes linearoligosaccharides into D-glucose.

Debranching enzymes which can attack amylopectin are divided into twoclasses: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41),respectively. Isoamylase hydrolyses alpha-1,6-D-glucosidic branchlinkages in amylopectin and beta-limit dextrins and can be distinguishedfrom pullulanases by the inability of isoamylase to attack pullulan, andby their limited action on alpha-limit dextrins.

It is well-known in the art to add isoamylases or pullulanases in starchconversion processes. Pullulanase is a starch debranching enzyme havingpullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzes thehydrolysis the α-1,6-glycosidic bonds in pullulan, releasing maltotriosewith reducing carbohydrate ends. Usually pullulanase is used incombination with an alpha amylase and/or a glucoamylase.

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules

Commercially, glucoamylases are used to convert starchy material, whichis already partially hydrolyzed by an alpha-amylase and e.g. apullulanase, to glucose in the form of syrup.

Before the enzymatic treatment the starch material, such as wholegrains, may be reduced in particle size, e.g., by milling, in order toopen up the structure and allowing for further processing. In drymilling whole kernels are milled and used. Wet milling gives a goodseparation of germ and meal (starch granules and protein) and is oftenapplied at locations where the starch hydrolyzate is used in theproduction of, e.g., syrups. Both dry and wet milling is well known inthe art of starch processing and may be used in a process of theinvention.

After milling typically the starch material is liquefied. Liquefactionis carried out in the presence of an alpha-amylase. During liquefaction,the long-chained starch is degraded into branched and linear shorterunits (maltodextrins) by the alpha-amylase.

For the production of glucose syrup, the liquefied starch material issaccharified. In a typical saccharification process, maltodextrinsproduced during liquefaction are converted into dextrose by adding aglucoamylase and a debranching enzyme, such as an isoamylase (U.S. Pat.No. 4,335,208) or a pullulanase. The temperature is lowered to 60° C.,prior to the addition of the glucoamylase and debranching enzyme. Thesaccharification process proceeds for 24-72 hours. Prior to addition ofthe saccharifying enzymes, the pH is reduced to below 4.5, whilemaintaining a high temperature (above 95° C.), to inactivate theliquefying alpha-amylase.

For the production of syrup, the enzyme compositions used should atleast comprise a glucoamylase and a pullulanase, however, oftenalpha-amylase activity will also be present, e.g. when using Aspergillusniger glucoamylase the A. niger alpha-amylase from the production hostwill also be present in the composition. Generally, the presence ofalpha-amylase is desirable because it increases the saccharificationspeed; however it also lowers the dextrose percentage (% DX) in thesachharification product, due to panose formation. Panose is atrisaccharide which is very slowly degraded by glucoamylase, andtherefore its formation negatively affects the peak % DX.

From practical and economic perspectives, one would prefer usingliquefied starch with a high percentage of dry solid (% DS) as startingmaterial in the saccharification reaction. Unfortunately, there is atrade-off between % DS and % DX: in order to arrive at saccharificationproducts with very high % DX, one will generally need to start fromliquefied starch with relatively low % DS. Optimizing the yield in termsof % DX is important for a number of purposes: for example, if thedesired end-product is crystalline dextrose, the yield of crystallinedextrose may be improved by up to 3% when the DX is increased by 1%.Even for purposes, where lower % DX is acceptable, it would still bedesirable to arrive at that % DX using liquefied starch with higher %DS.

Therefore, there is a need for improved technology for saccharificationof liquefied starch.

SUMMARY OF THE INVENTION

The invention provides compositions and processed for production ofimproved saccharification products in which it is possible to obtain ahigher glucose percentage/conversion rate despite even though thesaccharification process is performed using liquefied starch with a highdry solid content.

In particular, the invention provides a composition comprising aglucoamylase and a pullulanase, wherein the ratio of pullulanaseactivity expressed as NPUN/g to glucoamylase activity expressed as AGU/gis higher than 6.

The invention further provides a process for producing glucose syrupcomprising:

-   -   i) providing a composition comprising liquefied starch, and        adding to said composition a glucoamylase (AMG) and a        pullulanase;    -   ii) subjecting the composition to incubation under conditions        allowing starch hydrolysis/saccharification, and subsequently;    -   iii) adding an alpha-amylase to the said composition and        subjecting the composition to incubation under conditions        allowing starch hydrolysis/saccharification.

In addition, the invention provides a process for producing glucosesyrup from liquefied starch, comprising contacting the liquefied starchwith a composition according to the invention.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows the conversion rate (% DX) as function of time in acomparative study where liquefied starch was subjected tosaccharification using: (a) a conventional enzyme blend forsaccharification comprising glucoamylase, pullulanase and alpha-amylase;(b) a composition according to the present invention comprisingglucoamylase (0.25 AGU/gDS), Pullulanase (2.25 NPUN(X)/gDS, and noalpha-amylase; and (c) a composition according to the present inventioncomprising glucoamylase (0.25 AGU/gDS), Pullulanase (2.25 NPUN(X)/gDS,with addition of alpha-amylase ((0.0225 FAU(F)/gDS) at 24 hours.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Alpha-amylase: Alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, E.C.3.2.1.1) are a group of enzymes which catalyze the hydrolysis of starchand other linear and branched 1,4 glucosidic oligo- and polysaccharides.Alpha-amylases used according to the present invention may be obtainedfrom fungal or bacterial sources. For purposes of the present invention,fungal alpha amylase activity can be determined as FAU(A) using thealpha amylase assay described in the following paragraph. Activity ofbacterial alpha-amylases can be determined as Kilo Novo alpha-amylaseUnits (KNU) according to the procedure described in the paragraph “KiloNovo alpha-amylase Units (KNU)” below.

Acid alpha-Amylase Units (FAU(A)): Acid alpha-amylase activity may bemeasured in FAU(A) (Acid Fungal Alpha-amylase Units). 1 FAU(A) isdefined as the amount of enzyme which degrades 5.260 mg starch drymatter per hour under the standard conditions specified in the table“First reaction, starch degradation” below.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

FAU(A), the acid alpha-amylase activity is determined in accordance withthe following description. The principle of the reaction is based on thetwo steps. In the first step the enzyme acid alpha-amylase hydrolyzesstarch into different oligosaccharides. In the second step iodine formsa blue complex with starch but not with its degradation products. Theintensity of color is therefore directly proportional to theconcentration of starch. The activity is determined using reversecolorimetry as a reduction in the concentration of starch underspecified analytic conditions.

First reaction, starch degradation Substrate Starch, approx. 0.3 g/LBuffer Citrate, approx. 0.05M CaCl2, 1.85 mM pH 2.50 ± 0.05 Incubationtemperature 37° C. Reaction time 180 seconds Enzyme working range0.01-0.04 FAU(A)/mL Second reaction, starch-iodine complex Iodine 0.0432g/L Incubation temperature 37° C. Reaction time 60 seconds Wavelength600 nm

Kilo Novo Alpha-Amylase Units (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. The method is based on breakdown of starch in solution byamylase and the fact that starch gives a blue-black colour in presenceof iodine. As the enzyme reaction proceeds, aliquots of the reaction arewithdrawn and analyzed for their starch content by mixing with an iodinesolution. As starch is broken down, the blue-black colour in thepresence of iodine fades and gradually turns into a reddish-browncolour. This is compared with a coloured glass standard. The end pointis reached when the colour matches the glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions as defined in the “KNU” tablebelow (i.e., at 37° C.+/−0.05; 0.0003 M Ca2+; and pH 5.6) dextrinizes5260 mg/hour starch dry substance Merck Amylum solubile.

KNU Temperature 37 ± 0.05° C. pH 5.6 Substrate concentration 4.63 mg dryweight/mL Reaction time 7-20 min, up to 1 hr Ca²⁺ concentration approx.0.0003M for reaction mix containing 2 mL sample solution

Glucoamylase (AMG): The term glucoamylase (1,4-alpha-D-glucanglucohydrolase, EC 3.2.1.3) is defined as an enzyme, which catalyzes therelease of D-glucose from the non-reducing ends of starch or relatedoligo- and polysaccharide molecules. For purposes of the presentinvention, glucoamylase activity is determined as AGU according to theprocedure described in the following paragraphs.

Glucoamylase activity (AGU): The Glucoamylase Unit (AGU) is defined asthe amount of enzyme, which hydrolyzes 1 micromole maltose per minute ina 0.1 M acetate buffer at an incubation temperature 37° C., a pH of 4.3,a maltose starting concentration of 100 mM, and a reaction time of 6minutes, thereby generating alpha-D-glucose. The definition applies toan enzyme working range of 0.5-4.0 AGU/mL.

After incubation, the reaction may be stopped with NaOH and the amountsof glucose measured using the following two-step color reaction method:Glucose is phosphorylated by ATP, in a reaction catalyzed by hexokinase.The glucose-6-phosphate formed is oxidized to 6-phosphogluconate byglucose-6-phosphate dehydrogenase. In this same reaction, an equimolaramount of NAD+ is reduced to NADH with a resulting increase inabsorbance at 340 nm. Reaction conditions are as specified in the tablebelow:

Color reaction Tris approx. 35 mM ATP 0.7 mM NAD⁺ 0.7 mM Mg²⁺ 1.8 mMHexokinase >850 U/L Glucose-6-P-DH >850 U/L pH approx. 7.8 Temperature37.0° C. ± 1.0° C. Reaction time 420 sec Wavelength 340 nm

Pullulanase: The term “pullulanase” means a starch debranching enzymehaving pullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzesthe hydrolyses the α-1,6-glycosidic bonds in pullulan, releasingmaltotriose with reducing carbohydrate ends. For purposes of the presentinvention, pullulanase activity is determined as NPUN according to theprocedure described in the following paragraph.

Pullulanase activity (NPUN): The NPUN (New Pullulanase Unit Novozymes)is a unit of endopullulanase activity measured in the followingprocedure.

1 NPUN=One pullulanase unit (NPUN) is defined as the enzyme amount,which releases reducing ends equivalent to 0.35 μmol glucose per minuteunder the standard conditions specified in the table “First reaction,pullulan degradation” below.

In the first reaction, the substrate is equally present in both samplemain and sample blank. However, the reaction of sample main is performedat pH 5.0, while there is no reaction in the sample blank at pH 9.6,where neither pullulanases nor amyloglucosidases (glucoamylase) areenzymatically active.

First reaction, pullulan degradation Substrate BH4 reduced pullulan, 5.3g/L Buffer (main) Acetate, approx. 0.1M EDTA, 5.3 mM Acarbose, 0.018%(if sample contains glucoamylase) pH (main) 5.0 Buffer (blank) CHES, 42mM acetate, 17 mM EDTA, 5.3 mM pH (blank) 9.6 Incubation temperature 50°C. Reaction time 540 seconds Enzyme working range 0.03-0.15 NPUN/mL

In the second reaction, the pH is adjusted to approx. 9.6 and theglucose in samples is phosphorylated to non-reducingD-glucose-6-phosphate by glucokinase, which has optimal activity andstability in this range and is specific to glucose at pH 9 (ref. Goward,Biochem. J. 1986, 237, pp 415-420). This step depends on identical pH insample main and sample blank to remove equal amounts of glucose in both.

Second reaction, background glucose elimination Substrate glucose insample, after first reaction Buffer CHES, 58 mM (main) or 76 mM (blank)acetate, 43 mM (main) or 7.2 mM (blank) EDTA, 2.2 mM ATP, 1.11 mg/mlMgCl2, 4.4 mM Glucokinase 0.11 U/ml pH approx. 9.6 Incubationtemperature 50° C. Reaction time 720 seconds

The second reaction is stopped by and alkaline reagent >pH 11 containingPAHBAH (pHydroxy benzoic acid hydrazide) and bismuth, which complexeswith reducing sugars to produce color detected at 405 nm. The producedcolor is proportional to the pullulanase activity.

Third reaction, PAHBAH-Bi reaction Substrate maltotriose formed bypullulanase, after second reaction PAHBAH 56 mM Tartrate 75 mM Bi³⁺ 6.0mM NaOH 195 mM pH alkaline Incubation temperature 50° C. Reaction time1000 seconds Wavelength 405 nm

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. According to some embodiments, themature polypeptide of SEQ ID NO: 2 consists essentially of amino acids18 to 573 of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3consists essentially of amino acids 18 to 573 of SEQ ID NO: 3, themature polypeptide of SEQ ID NO: 4 consists essentially of amino acids18 to 573 of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5consists essentially of amino acids 18 to 573 of SEQ ID NO: 5, themature polypeptide of SEQ ID NO: 6 consists essentially of amino acids18 to 573 of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 7consists essentially of amino acids 18 to 573 of SEQ ID NO: 7, themature polypeptide of SEQ ID NO: 8 consists essentially of amino acids18 to 573 of SEQ ID NO: 8, the mature polypeptide of SEQ ID NO: 9consists essentially of amino acids 18 to 576 of SEQ ID NO: 9, themature polypeptide of SEQ ID NO: 10 consists essentially of amino acids18 to 576 of SEQ ID NO: 10, the mature polypeptide of SEQ ID NO: 11consists essentially of amino acids 21 to 618 of SEQ ID NO: 11, themature polypeptide of SEQ ID NO: 12 consists essentially of amino acids1 to 559 of SEQ ID NO: 12, the mature polypeptide of SEQ ID NO: 13consists essentially of amino acids 1 to 928 of SEQ ID NO: 13, themature polypeptide of SEQ ID NO: 14 consists essentially of amino acids1 to 828 of SEQ ID NO: 14, the mature polypeptide of SEQ ID NO: 15consists essentially of amino acids 1 to 928 of SEQ ID NO: 15, themature polypeptide of SEQ ID NO: 16 consists essentially of amino acids1 to 928 of SEQ ID NO: 16, the mature polypeptide of SEQ ID NO: 17consists essentially of amino acids 1 to 484 of SEQ ID NO: 17, themature polypeptide of SEQ ID NO: 18 consists essentially of amino acids1 to 583 of SEQ ID NO: 18, the mature polypeptide of SEQ ID NO: 19consists essentially of amino acids 22 to 471 of SEQ ID NO: 19, themature polypeptide of SEQ ID NO: 20 consists essentially of amino acids1 to 514 of SEQ ID NO: 20, the mature polypeptide of SEQ ID NO: 21consists essentially of amino acids 1 to 483 of SEQ ID NO: 21, themature polypeptide of SEQ ID NO: 22 consists essentially of amino acids1 to 483 of SEQ ID NO: 22, the mature polypeptide of SEQ ID NO: 23consists essentially of amino acids 1 to 483 of SEQ ID NO: 23, themature polypeptide of SEQ ID NO: 24 consists essentially of amino acids1 to 483 of SEQ ID NO: 24, the mature polypeptide of SEQ ID NO: 25consists essentially of amino acids 1 to 483 of SEQ ID NO: 25, themature polypeptide of SEQ ID NO: 26 consists essentially of amino acids21 to 607 of SEQ ID NO: 26, and/or the mature polypeptide of SEQ ID NO:27 consists essentially of amino acids 19 to 640 of SEQ ID NO: 27.

In further embodiments, the mature polypeptide of SEQ ID NO: 2 consistsof amino acids 18 to 573 of SEQ ID NO: 2, the mature polypeptide of SEQID NO: 3 consists of amino acids 18 to 573 of SEQ ID NO: 3, the maturepolypeptide of SEQ ID NO: 4 consists of amino acids 18 to 573 of SEQ IDNO: 4, the mature polypeptide of SEQ ID NO: 5 consists of amino acids 18to 573 of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6 consistsof amino acids 18 to 573 of SEQ ID NO: 6, the mature polypeptide of SEQID NO: 7 consists of amino acids 18 to 573 of SEQ ID NO: 7, the maturepolypeptide of SEQ ID NO: 8 consists of amino acids 18 to 573 of SEQ IDNO: 8, the mature polypeptide of SEQ ID NO: 9 consists of amino acids 18to 576 of SEQ ID NO: 9, the mature polypeptide of SEQ ID NO: 10 consistsof amino acids 18 to 576 of SEQ ID NO: 10, the mature polypeptide of SEQID NO: 11 consists of amino acids 21 to 618 of SEQ ID NO: 11, the maturepolypeptide of SEQ ID NO: 12 consists of amino acids 1 to 559 of SEQ IDNO: 12, the mature polypeptide of SEQ ID NO: 13 consists of amino acids1 to 928 of SEQ ID NO: 13, the mature polypeptide of SEQ ID NO: 14consists essentially of amino acids 1 to 828 of SEQ ID NO: 14, themature polypeptide of SEQ ID NO: 15 consists of amino acids 1 to 928 ofSEQ ID NO: 15, the mature polypeptide of SEQ ID NO: 16 consists of aminoacids 1 to 928 of SEQ ID NO: 16, the mature polypeptide of SEQ ID NO: 17consists of amino acids 1 to 484 of SEQ ID NO: 17, the maturepolypeptide of SEQ ID NO: 18 consists of amino acids 1 to 583 of SEQ IDNO: 18, the mature polypeptide of SEQ ID NO: 19 consists of amino acids22 to 471 of SEQ ID NO: 19, the mature polypeptide of SEQ ID NO: 20consists of amino acids 1 to 514 of SEQ ID NO: 20, the maturepolypeptide of SEQ ID NO: 21 consists of amino acids 1 to 483 of SEQ IDNO: 21, the mature polypeptide of SEQ ID NO: 22 consists of amino acids1 to 483 of SEQ ID NO: 22, the mature polypeptide of SEQ ID NO: 23consists of amino acids 1 to 483 of SEQ ID NO: 23, the maturepolypeptide of SEQ ID NO: 24 consists of amino acids 1 to 483 of SEQ IDNO: 24, the mature polypeptide of SEQ ID NO: 25 consists of amino acids1 to 483 of SEQ ID NO: 25, the mature polypeptide of SEQ ID NO: 26consists of amino acids 21 to 607 of SEQ ID NO: 26, the maturepolypeptide of SEQ ID NO: 27 consists of amino acids 19 to 640 of SEQ IDNO: 27.

The prediction of mature polypeptide sequences may be based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 17 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9and SEQ ID NO: 10 are a signal peptide. Likewise, amino acids 1 to 20 ofSEQ ID NO: 11, amino acids 1 to 21 of SEQ ID NO: 19, amino acids 1 to 20of SEQ ID NO: 26, and amino acids 1-18 of SEQ ID NO: 27 are predicted tobe signal peptides.

The sequence defined by amino acids 19 to 474 (particularly 19 to 471)of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, or SEQ ID NO: 8 or amino acids 19 to 471 of SEQ ID NO: 9or of SEQ ID NO: 10 is the catalytic domain. The sequence defined byamino acids 480 to 573 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or amino acids 483to 576 of SEQ ID NO: 9 or SEQ ID NO: 10 is a starch binding domain.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Degree of polymerization (DP): DP refers to the number (n) ofanhydroglucopyranose units in a given saccharide. Examples of DP1 aremonosaccharides, such as glucose and fructose. DP2 are disaccharides,such as maltose and sucrose.

DS and % DS: As used herein, “DS” is an abbreviation of the term “DrySolid”. In the context of the present invention the solid content ofliquefied starch (including starch slurry or partially hydrolysedstarch) is provided as percentage of dry solid (% DS) unless otherwisestated. The percentage of dry solid is calculated based on weight (w/w%).

% DX: In the context of the present invention dextrose percentage (% DX)is used as a measure of the amounts of dextrose obtained duringsaccharification or after completion of the saccharification process.The dextrose percentage is calculated based on weight (w/w %).

Aspects and Embodiments of the Invention

The present inventors have surprisingly found that delayed or stagedaddition of alpha-amylase during saccharification of liquefied starchhas several benefits: the positive effect of the alpha-amylase onsaccharification speed is maintained and, in addition, the peak % DX ismuch improved. This beneficial effect is observed even when thealpha-amylase is added at a relatively late stage in thesaccharification process. In addition, the inventors have found thatincreasing the ratio of pullulanase to glucoamylase in the enzyme blendadded at onset of the saccharification process also aids in arriving ata higher % DX in the saccharification product.

In a first aspect the present invention provides a compositioncomprising a glucoamylase and a pullulanase, wherein the ratio ofpullulanase activity expressed as NPUN/g to glucoamylase activityexpressed as AGU/g is higher than 6, such as higher than 7, higher than8, higher than 9 or within the range of 6-20, such as within the rangeof 6-19, within the range of 6-18. within the range of 6-17, within therange of 6-16, within the range of 6-15, within the range of 6-14,within the range of 6-13, within the range of 6-12, within the range of6-11, within the range of 7-20, within the range of 7-19, within therange of 7-18, within the range of 7-17, within the range of 7-16,within the range of 7-15, within the range of 7-14, within the range of7-13, within the range of 7-12, within the range of 7-11, within therange of 8-20, within the range of 8-19, within the range of 8-18,within the range of 8-17, within the range of 8-16, within the range of8-15, within the range of 8-14, within the range of 8-13, within therange of 8-12, within the range of 8-11, within the range of 9-20,within the range of 9-19, within the range of 9-18, within the range of9-17, within the range of 9-16, within the range of 9-15, within therange of 9-14, within the range of 9-13, within the range of 9-12 orsuch as within the range of 9-11.

It is to be understood that when delaying the addition of alpha amylaseduring saccharification, it is desirable that the composition comprisingglucoamylase and pullulanase which is added at onset of thesaccharification contains no or at least very little alpha-amylaseactivity: Hence, in some embodiments of the invention the compositioncomprises no alpha-amylase activity. Alternatively, if said compositiondoes comprise a polypeptide having alpha-amylase activity and thatpolypeptide is of fungal origin, then the ratio of glucoamylase activityexpressed as AGU/g to alpha-amylase activity expressed as Fungalalpha-Amylase Units FAU(A)/g is higher than 29, such as higher than 50,higher than 100, higher than 200, higher than 300, higher than 400,higher than 500, higher than 600, higher than 800, higher than 1000,higher than 2000, higher than 3000, higher than 4000 or such as higherthan 5000. If the polypeptide having alpha-amylase activity is ofbacterial origin, then the ratio of glucoamylase activity expressed asAGU/g to alpha-amylase activity expressed as Kilo Novo alpha-amylaseUnits (KNU)/g is higher than 29, such as higher than 50, higher than100, higher than 200, higher than 300, higher than 400, higher than 500,higher than 600, higher than 800, higher than 1000, higher than 2000,higher than 3000, higher than 4000 or such as higher than 5000.

In some specific embodiments, the invention provides a composition,wherein the ratio of pullulanase activity expressed as NPUN/g toglucoamylase activity expressed as AGU/g is higher than 6, and whereinif said composition comprises a polypeptide having alpha-amylaseactivity and that polypeptide is of fungal origin, then the ratio ofglucoamylase activity expressed as AGU/g to alpha-amylase activityexpressed as Fungal alpha-Amylase Units FAU(A)/g is higher than 100, Ifthe polypeptide having alpha-amylase activity is of bacterial origin,then the ratio of glucoamylase activity expressed as AGU/g toalpha-amylase activity expressed as Kilo Novo alpha-amylase Units(KNU)/g is higher than 100.

In other specific embodiments, the invention provides a composition,wherein the ratio of pullulanase activity expressed as NPUN/g toglucoamylase activity expressed as AGU/g is within the range of 6-20,and wherein if said composition comprises a polypeptide havingalpha-amylase activity and that polypeptide is of fungal origin, thenthe ratio of glucoamylase activity expressed as AGU/g to alpha-amylaseactivity expressed as Fungal alpha-Amylase Units FAU(A)/g is higher than100, If the polypeptide having alpha-amylase activity is of bacterialorigin, then the ratio of glucoamylase activity expressed as AGU/g toalpha-amylase activity expressed as Kilo Novo alpha-amylase Units(KNU)/g is higher than 100.

In further specific embodiments, the invention provides a composition,wherein the ratio of pullulanase activity expressed as NPUN/g toglucoamylase activity expressed as AGU/g is within the range of 6-20,and wherein if said composition comprises a polypeptide havingalpha-amylase activity and that polypeptide is of fungal origin, thenthe ratio of glucoamylase activity expressed as AGU/g to alpha-amylaseactivity expressed as Fungal alpha-Amylase Units FAU(A)/g is higher than500. If the polypeptide having alpha-amylase activity is of bacterialorigin, then the ratio of glucoamylase activity expressed as AGU/g toalpha-amylase activity expressed as Kilo Novo alpha-amylase Units(KNU)/g is higher than 500.

In even further specific embodiments, the invention provides acomposition, wherein the ratio of pullulanase activity expressed asNPUN/g to glucoamylase activity expressed as AGU/g is within the rangeof 7-15, and wherein if said composition comprises a polypeptide havingalpha-amylase activity and that polypeptide is of fungal origin, thenthe ratio of glucoamylase activity expressed as AGU/g to alpha-amylaseactivity expressed as Fungal alpha-Amylase Units FAU(A)/g is higher than100. If the polypeptide having alpha-amylase activity is of bacterialorigin, then the ratio of glucoamylase activity expressed as AGU/g toalpha-amylase activity expressed as Kilo Novo alpha-amylase Units(KNU)/g is higher than 100.

Other specific embodiments of the invention provides a composition,wherein the ratio of pullulanase activity expressed as NPUN/g toglucoamylase activity expressed as AGU/g is within the range of 7-15,and wherein if said composition comprises a polypeptide havingalpha-amylase activity and that polypeptide is of fungal origin, thenthe ratio of glucoamylase activity expressed as AGU/g to alpha-amylaseactivity expressed as Fungal alpha-Amylase Units FAU(A)/g is higher than500. If the polypeptide having alpha-amylase activity is of bacterialorigin, then the ratio of glucoamylase activity expressed as AGU/g toalpha-amylase activity expressed as Kilo Novo alpha-amylase Units(KNU)/g is higher than 500.

A glucoamylase included in the compositions or used in the processesaccording to the present invention may be derived from any suitablesource, e.g., derived from a microorganism or a plant. Preferredglucoamylases are of fungal or bacterial origin, selected from the groupconsisting of Aspergillus glucoamylases, in particular A. niger G1 or G2glucoamylase (Boel et al., 1984, EMBO J. 3 (5): 1097-1102), or variantsthereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO01/04273 (from Novozymes, Denmark); the A. awamori glucoamylasedisclosed in WO 84/02921, A. oryzae glucoamylase (Agric. Biol. Chem.,1991, 55 (4): 941-949), or variants or fragments thereof. OtherAspergillus glucoamylase variants include variants with enhanced thermalstability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505);D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chenet al., 1994, Biochem. J. 301: 275-281); disulphide bonds, A246C(Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction ofPro residues in position A435 and S436 (Li et al., 1997, Protein Eng.10: 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 andNagasaka et al., 1998, “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, andTalaromyces thermophilus (U.S. Pat. No. 4,587,215).

Contemplated fungal glucoamylases include Trametes cingulata, disclosedin WO 2006/069289.

In an embodiment, the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus as described in WO2011/066576 (SEQ ID NOs 2, 4 or 6), or from a strain of the genusGloeophyllum, in particular a strain of Gloeophyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) or a strain of thegenus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed inWO 2012/064351 (SEQ ID NO: 2) (all references hereby incorporated byreference) or a strain of Penicillium, in particular Penicilliumoxalicum disclosed in WO2011/127802 (SEQ ID NO: 2) or WO2013/036526.Contemplated are also glucoamylases which exhibit a high identity to anyof the above-mentioned glucoamylases, i.e., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to any oneof the mature parts of the enzyme sequences mentioned above.

In an embodiment, the glucoamylase is derived from a strain of the genusTrichoderma, in particular as described in WO2009/048487, WO2009/048488,WO2008/045489, WO2011/022465, WO2012/001139.

Commercially available glucoamylase compositions include AMG 200L; AMG300L; SANT™ SUPER, SANT™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL,SPIRIZYME™ B4U, SPIRIZYME ULTRA™, SPIRIZYME EXCEL™ and AMG™ E (fromNovozymes NS, Denmark); OPTIDEX™ 300, GC480™ and GC147™ (from GenencorInt., USA); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™and G990 ZR (from DuPont-Genencor)

According to specific embodiments, the said glucoamylase is selectedfrom the group consisting of an Aspergillus niger glucoamylase, aTalaromyces emersonii glucoamylase, a Trametes cingulata glucoamylase aGloeophyllum trabeum glucoamylase, a Trichoderma Reesei glucoamylase, aHumicola insolens glucoamylase and an Aspergillus fumigatusglucoamylase, and hybrids and variants thereof.

In principle, any pullulanase may be used in a process of the presentinvention. In an embodiment, the pullulanase is a pullulanase fromBacillus deramificans, e.g., disclosed in U.S. Pat. No. 6,074,854 andU.S. Pat. No. 5,817,498, or a pullulanase derived from Bacillusacidopullulyticus, e.g., disclosed in WO2009/075682 (SEQ ID 4; GENESEQP:AXB71624). The pullulanase may also be a hybrid or a variant of any ofthese pullulanases.

Commercially available pullulanases include Promozyme D2 available fromNovozymes NS, Bagsvaerd, Denmark), Novozym 26062 (Novozymes) and OptimaxL 1000 (DuPont-Genencor).

The composition according to any of the preceding claims, wherein thepullulanase comprises/consists essentially of/consists of an amino acidsequence selected from the group consisting of

-   -   i) The amino acid sequence set forth in any one of SEQ ID NOs:        13-16 or a mature polypeptide thereof;    -   ii) A subsequence of the amino acid sequence set forth in any        one of SEQ ID NO: 13-16 or of said mature polypeptide thereof;    -   iii) A variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in i) and ii);

wherein when said pullulanase comprises a subsequence as defined in ii)or a variant amino acid sequence as defined in iii), it preferably hasat least 80%, such as at least 90% of the pullulanase activity of therespective amino acid defined in i) of which it is a subsequence orvariant, when tested as set forth above in relation to the definition of“Pullulanase activity (NPUN)”.

In the composition according to the invention, the glucoamylase may inparticular comprise an amino acid sequence selected from the groupconsisting of:

-   -   i) The amino acid sequence set forth in any one of SEQ ID NOs:        2-12 and 27 or a mature polypeptide thereof;    -   ii) A subsequence of the amino acid sequence set forth in any        one of SEQ ID NOs: 2-12 and 27 or of said mature polypeptide        thereof;    -   iii) A variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in i) and ii)

wherein when said glucoamylase comprises a subsequence as defined in ii)or an amino acid sequence as defined in iii), it preferably has at least80%, such as at least 90% of the glucoamylase activity of the respectiveamino acid defined in i) of which it is a subsequence or variant, whentested as set forth above in relation to the definition of “Glucoamylaseactivity (AGU)”.

In particular, the composition according to the invention may comprise:

-   -   i) a glucoamylase comprising an amino acid sequence selected        from the group consisting of the amino acid sequence set forth        in SEQ ID NO: 12 or a mature polypeptide thereof,    -   ii) a subsequence of the amino acid sequence set forth in SEQ ID        NO: 12 or of said mature polypeptide thereof, and    -   iii) an amino acid sequence, which has at least 70%, such as at        least 75%, at least 80%, at least 85%, at least 90%, at least        91%, at least 92%, at least 93%, at least 94%, at least 95%, at        least 96%, at least 97%, at least 98%, at least 99%, such as at        least 99.5% sequence identity to any one of the amino acids        sequences set forth in i) and ii);    -   wherein when said glucoamylase is a subsequence as defined        in ii) or a variant amino acid sequence as defined in iii), it        preferably has at least 80%, such as at least 90% of the        glucoamylase activity of the respective amino acid defined in i)        of which it is a subsequence or variant, when tested as set        forth above in relation to the definition of “Glucoamylase        activity (AGU)”.

In combination with

-   -   iv) a pullulanase comprising an amino acid sequence selected        from the group consisting of the amino acid sequence set forth        in any one of SEQ ID NOs: 13-16 or a mature polypeptide thereof,    -   v) a subsequence of the amino acid sequence set forth in any one        of SEQ ID NOs: 13-16 or of said mature polypeptide thereof, and    -   vi) an amino acid sequence, which has at least 70%, such as at        least 75%, at least 80%, at least 85%, at least 90%, at least        91%, at least 92%, at least 93%, at least 94%, at least 95%, at        least 96%, at least 97%, at least 98%, at least 99%, such as at        least 99.5% sequence identity to any one of the amino acids        sequences set forth in iv) and v);    -   wherein when said pullulanase comprises is a subsequence as        defined in v) or a variant amino acid sequence as defined in        vi), it preferably has at least 80%, such as at least 90% of the        pullulanase activity of the respective amino acid defined in iv)        of which it is a subsequence or variant, when tested as set        forth above in relation to the definition of “Pullulanase        activity (NPUN)”.

In related embodiments, the glucoamylase consists essentially of anamino acid sequence as set forth in any of items i)-iii) above. In otherrelated embodiments/consists the glucoamylase consists of an amino acidsequence as set forth in any of items i)-iii) above.

In the composition according to any of the preceding claims, thealpha-amylase may comprise an amino acid sequence selected from thegroup consisting of

-   -   i) The amino acid sequence set forth in any one of SEQ ID NOs: 1        and 17-26 or a mature polypeptide thereof;    -   ii) A subsequence of the amino acid sequence set forth in any        one of SEQ ID NOs: 1 and 17-26 or of said mature polypeptide        thereof;    -   iii) An amino acid sequence, which has at least 70%, such as at        least 75%, at least 80%, at least 85%, at least 90%, at least        91%, at least 92%, at least 93%, at least 94%, at least 95%, at        least 96%, at least 97%, at least 98%, at least 99%, such as at        least 99.5% sequence identity to any one of the amino acids        sequences set forth in i) and ii).

When the alpha-amylase defined above is a subsequence or a variant, itpreferably has at least 80%, such as at least 90% of the alpha-amylaseactivity of the respective alpha-amylase selected from SEQ ID NOs: 1 and17-26 or the mature polypeptide thereof of which it is a subsequence orvariant, when tested as set forth above in relation to the definition of“Acid alpha-Amylase Units (FAU(A))” or “Alpha-amylase Activity (KNU)”:In the present context an alpha-amylase comprising an amino acidsequences selected from the group consisting of SEQ ID NOs: 1, 17-19 and26 or the mature polypeptide thereof, is considered to be a fungal alphaamylase and activity is tested as provided in relation to the abovedefinition of “Acid alphaAmylase Units (FAU(A))”. An alpha-amylasecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 20-25 or the mature polypeptide thereof is considered to bea bacterial alpha amylase and activity is tested as provided in relationto the above definition of “Kilo Novo alpha-amylase Units (KNU)”.

In related embodiments, the alpha-amylase consists essentially of anamino acid sequence as set forth in any of items i)-iii) above. In otherrelated embodiments the alpha-amylase consists of an amino acid sequenceas set forth in any of items i)-iii) above.

The composition provided according to the invention may be a liquid orsolid composition. In currently preferred embodiments it is a liquidcomposition.

Another aspect of the invention provides a process for producing glucosesyrup. In the process according to the invention the addition of alphaamylase is staged in the sense that glucoamylase and pullulanase isadded at onset of the saccharification reaction, while the addition ofalpha-amylase is postponed at least until a significant or substantialpart of the liquefied starch has been saccharified. Hence, the processof the invention comprises,

-   -   i) providing a composition comprising liquefied starch, and        adding to said composition a glucoamylase (AMG) and a        pullulanase;    -   ii) subjecting the composition to incubation under conditions        allowing starch hydrolysis/saccharification, and subsequently    -   iii) adding an alpha-amylase to the said composition and        subjecting the composition to incubation under conditions        allowing starch hydrolysis/saccharification.

The amounts of glucoamylase added in step i) of the process may inparticular correspond to 0.05-0.5 AGU/gDS, such as 0.05-0.4 AGU/gDS,0.05-0.35 AGU/gDS, 0.05-0.3 AGU/gDS, 0.075-0.5 AGU/gDS, 0.075-0.4AGU/gDS, 0.075-0.35 AGU/gDS 0.075-0.3 AGU/gDS, 0.075-0.5 AGU/gDS 0.1-0.5AGU/gDS, 0.1-0.4 AGU/gDS, 0.1-0.35 AGU/gDS, 0.1-0.3 AGU/gDS, 0.125-0.5AGU/gDS, 0.125-0.4 AGU/gDS, 0.125-0.35 AGU/gDS, 0.125-0.3 AGU/gDS,0.15-0.5 AGU/gDS, 0.15-0.4 AGU/gDS, 0.15-0.35 AGU/gDS, 0.15-0.3 AGU/gDS,0.175-0.5 AGU/gDS, 0.175-0.4 AGU/gDS, 0.175-0.35 AGU/gDS, 0.175-0.3AGU/gDS, 0.2-0.5 AGU/gDS, 0.2-0.4 AGU/gDS, 0.2-0.35 AGU/gDS, 0.2-0.3AGU/gDS, 0.24-0.5 AGU/gDS, 0.24-0.4 AGU/gDS, 0.24-0.35 AGU/gDS or suchas 0.24-0.26 AGU/gDS.

The amounts of pullulanase added in step i) of the process may inparticular correspond to 0.05-5 NPUN(X)/gDS, such as 0.05-4.5NPUN(X)/gDS, 0.05-4 NPUN(X)/gDS, 0.05-3.5 NPUN(X)/gDS, 0.05-3NPUN(X)/gDS, 0.05-2.5 NPUN(X)/gDS, 0.1-5 NPUN(X)/gDS, 0.1-4 NPUN(X)/gDS,0.1-3.5 NPUN(X)/gDS, 0.1-3 NPUN(X)/gDS, 0.1-2.5 NPUN(X)/gDS, 0.25-5NPUN(X)/gDS, 0.25-4.5 NPUN(X)/gDS, 0.25-4 NPUN(X)/gDS, 0.25-3.5NPUN(X)/gDS, 0.25-3 NPUN(X)/gDS, 0.5-5 NPUN(X)/gDS, 0.5-4.5 NPUN(X)/gDS,0.5-4 NPUN(X)/gDS, 0.5-3.5 NPUN(X)/gDS, 0.05-3 NPUN(X)/gDS, 0.5-2.5NPUN(X)/gDS, 1.0-5 NPUN(X)/gDS, 1.0-4.5 NPUN(X)/gDS, 1.0-4 NPUN(X)/gDS,1.0-3.5 NPUN(X)/gDS, 1.0-3 NPUN(X)/gDS, 1.0-2.5 NPUN(X)/gDS, 1.5-5NPUN(X)/gDS, 1.5-4.5 NPUN(X)/gDS, 1.5-4 NPUN(X)/gDS, 1.5-3.5NPUN(X)/gDS, 1.5-3 NPUN(X)/gDS, 1.5-2.5 NPUN(X)/gDS, 2-5 NPUN(X)/gDS,2-4.5 NPUN(X)/gDS, 2-4 NPUN(X)/gDS, 2-3.5 NPUN(X)/gDS, 2-3 NPUN(X)/gDS,2-5 NPUN(X)/gDS, 2-2.5 NPUN(X)/gDS, 2.25-5 NPUN(X)/gDS, 2.25-4.5NPUN(X)/gDS, 2.25-4 NPUN(X)/gDS, 2.25-3.5 NPUN(X)/gDS, 2.25-3NPUN(X)/gDS, 2.25-2.5 NPUN(X)/gDS, or such as 2.50-2.54 NPUN(X)/gDS.

Where the alpha-amylase added in step iii) of the process according tothe invention is a fungal alpha-amylase, the amounts may in particularcorrespond to 0.0005-0.025 FAU(A)/gDS, such as 0.0005-0.0225 FAU(A)/gDS,0.0005-0.02 FAU(A)/gDS, 0.0005-0.0175 FAU(A)/gDS, 0.0005-0.015FAU(A)/gDS, 0.0005-0.0125 FAU(A)/gDS, 0.001-0.025 FAU(A)/gDS,0.001-0.0225 FAU(A)/gDS, 0.001-0.02 FAU(A)/gDS, 0.001-0.0175 FAU(A)/gDS,0.001-0.015 FAU(A)/gDS, 0.001-0.0125 FAU(A)/gDS, 0.0025-0.025FAU(A)/gDS, 0.0025-0.0225 FAU(A)/gDS, 0.0025-0.02 FAU(A)/gDS,0.0025-0.0175 FAU(A)/gDS, 0.0025-0.015 FAU(A)/gDS, 0.0025-0.0125FAU(A)/gDS, 0.005-0.025 FAU(A)/gDS, 0.005-0.0225 FAU(A)/gDS, 0.005-0.02FAU(A)/gDS, 0.005-0.0175 FAU(A)/gDS, 0.005-0.015 FAU(A)/gDS,0.005-0.0125 FAU(A)/gDS, 0.0075-0.025 FAU(A)/gDS, 0.0075-0.0225FAU(A)/gDS, 0.0075-0.02 FAU(A)/gDS, 0.0075-0.0175 FAU(A)/gDS,0.0075-0.015 FAU(A)/gDS, 0.0075-0.0125 FAU(A)/gDS, 0.008-0.025FAU(A)/gDS, 0.008-0.0225 FAU(A)/gDS, 0.008-0.02 FAU(A)/gDS, 0.008-0.0175FAU(A)/gDS, 0.008-0.015 FAU(A)/gDS, 0.008-0.0125 FAU(A)/gDS, 0.009-0.01FAU(A)/gDS or such as 0.0095 FAU(A)/gDS.

Where the alpha-amylase added in step iii) of the process according tothe invention is a bacterial alpha-amylase, the amounts may inparticular correspond to 0.0005-0.025 KNU/gDS, such as 0.0005-0.0225KNU/gDS, 0.0005-0.02 KNU/gDS, 0.0005-0.0175 KNU/gDS, 0.0005-0.015KNU/gDS, 0.0005-0.0125 KNU/gDS, 0.001-0.025 KNU/gDS, 0.001-0.0225KNU/gDS, 0.001-0.02 KNU/gDS, 0.001-0.0175 KNU/gDS, 0.001-0.015 KNU/gDS,0.001-0.0125 KNU/gDS, 0.0025-0.025 KNU/gDS, 0.0025-0.0225 KNU/gDS,0.0025-0.02 KNU/gDS, 0.0025-0.0175 KNU/gDS, 0.0025-0.015 KNU/gDS,0.0025-0.0125 KNU/gDS, 0.005-0.025 KNU/gDS, 0.005-0.0225 KNU/gDS,0.005-0.02 KNU/gDS, 0.005-0.0175 KNU/gDS, 0.005-0.015 KNU/gDS,0.005-0.0125 KNU/gDS, 0.0075-0.025 KNU/gDS, 0.0075-0.0225 KNU/gDS,0.0075-0.02 KNU/gDS, 0.0075-0.0175 KNU/gDS, 0.0075-0.015 KNU/gDS,0.0075-0.0125 KNU/gDS, 0.008-0.025 KNU/gDS, 0.008-0.0225 KNU/gDS,0.008-0.02 KNU/gDS, 0.008-0.0175 KNU/gDS, 0.008-0.015 KNU/gDS,0.008-0.0125 KNU/gDS, 0.009-0.01 KNU/gDS or such as 0.0095 KNU/gDS.

In some embodiments, the invention provides a process, wherein theamounts of glucoamylase added in step i) of the process correspond to0.05-0.5 AGU/gDS, the amounts of pullulanase added in step i) of theprocess correspond to 0.05-5 NPUN(X)/gDS, and the amounts of fungalalpha-amylase added in step iii) of the process correspond to0.0005-0.025 (FAU)(A)/gDS or the amounts of bacterial alpha-amylaseadded in step iii) of the process correspond to 0.0005-0.025 KNU/gDS.

In still other embodiments, the invention provides a process, whereinthe amounts of glucoamylase added in step i) of the process correspondto 0.175-0.3 AGU/gDS, the amounts of pullulanase added in step i) of theprocess correspond to 2-4 NPUN(X)/gDS, and the amounts of alpha-amylaseadded in step iii) of the process correspond to 0.0025-0.02 (FAU)(A)/gDSor the amounts of bacterial alpha-amylase added in step iii) of theprocess correspond to 0.0025-0.02 KNU/gDS.

In further embodiments, the invention pertains to a process, wherein theamounts of glucoamylase added in step i) of the process correspond to0.2-0.3 AGU/gDS, the amounts of pullulanase added in step i) of theprocess correspond to 2-3 NPUN(X)/gDS, and the amounts of alpha-amylaseadded in step iii) of the process correspond to 0.008-0.0125(FAU)(A)/gDS or the amounts of bacterial alpha-amylase added in stepiii) of the process correspond to 0.008-0.0125 KNU/gDS.

When delaying the addition of alpha amylase during saccharification, itis desirable that no or at least very little alpha-amylase activity isadded together with the glucoamylase and the pullulanase at onset of thesaccharification process. Therefore, in some embodiments the processcomprising adding to said composition comprising liquefied starch aformulation comprising a glucoamylase and a pullulanase, wherein if saidformulation comprises a polypeptide having alpha-amylase activity, andthat polypeptide is of bacterial origing, then the ratio of glucoamylaseactivity expressed as AGU/g to alpha-amylase activity expressed asFungal alphaAmylase Units FAU(A)/g in said composition is higher than29, such as higher than 50, higher than 100, higher than 200, higherthan 300, higher than 400, higher than 500, higher than 600, higher than800, higher than 1000, higher than 2000, higher than 3000, higher than4000 or such as higher than 5000. If the polypeptide havingalpha-amylase activity is of bacterial origin, then the ratio ofglucoamylase activity expressed as AGU/g to alpha-amylase activityexpressed as Kilo Novo alpha-amylase Units (KNU)/g is higher than 29,such as higher than 50, higher than 100, higher than 200, higher than300, higher than 400, higher than 500, higher than 600, higher than 800,higher than 1000, higher than 2000, higher than 3000, higher than 4000or such as higher than 5000.

According to some embodiments, the glucoamylase and the pullulanaseadded in step ii) of the process are in a composition according to theinvention as defined in the above.

The incubation in step ii) of the process may have a duration of from2-35 hours, such as from 2-30 hours, from 2-28 hours, from 2-26 hours,from 2-24, from 2-22 hours, from 2-20 hours, from 2-18 hours, from 2-16hours, from 2-14 hours, from 2-12 hours, from 2-10 hours, from 2-8hours, from 4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26hours, from 4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours,from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10 hours, from4-8 hours, from 10-35 hours, from 10-30 hours, from 10-28 hours, from10-26 hours, from 10-24 hours, from 10-22 hours, from 10-20 hours, from10-18 hours, from 10-16 hours, from 10-14 hours, from 10-12 hours, from14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16hours, from 18-35 hours, from 18-30 hours, from 18-28 hours, from 18-26hours, from 18-24, from 18-22 hours, from 18-20 hours, from 20-35 hours,from 20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from20-22 hours, form 22-35 hours, from 22-30 hours, from 22-28 hours, from22-26 hours, from 22-24 hours or such or sus as a duration of 24 hours.

The incubation in step iii) of the process may have a duration of from2-94 hours, such as from 2-90 hours, from 2-80 hours, from 2-70 hours,from 2-60, from 2-55 hours, from 2-50 hours, from 2-45 hours, from 2-40hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from 2-26hours, from 2-24, from 2-22 hours, from 2-20 hours, from 2-18 hours,from 2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10 hours, from2-8 hours, from 4-94 hours, from 4-90 hours, from 4-80 hours, from 4-70hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45 hours,from 4-40 hours, from 4-35 hours, from 4-30 hours, from 4-28 hours, from4-26 hours, from 4-24, from 4-22 hours, from 4-20 hours, from 4-18hours, from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10hours, from 4-8 hours, from 10-94 hours, from 10-90 hours, from 10-80hours, from 10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,from 10-28 hours, from 10-26 hours, from 10-24, from 10-22 hours, from10-20 hours, from 10-18 hours, from 10-16 hours, from 10-14 hours, from10-12 hours, from 14-94 hours, from 14-90 hours, from 14-80 hours, from14-70 hours, from 14-60, from 14-55 hours, from 14-50 hours, from 14-45hours, from 14-40 hours, from 14-35 hours, from 14-30 hours, from 14-28hours, from 14-26 hours, from 14-24, from 14-22 hours, from 14-20 hours,from 14-18 hours, from 14-16 hours, from 20-94 hours, from 20-90 hours,from 20-80 hours, from 20-70 hours, from 20-60, from 20-55 hours, from20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours, from20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from 20-22hours, from 24-94 hours, from 24-90 hours, from 24-80 hours, from 24-70hours, from 24-60, from 24-55 hours, from 24-50 hours, from 24-45 hours,from 24-40 hours, from 24-35 hours, from 24-30 hours, from 24-28 hours,from 24-26 hours, from 30-94 hours, from 30-90 hours, from 30-80 hours,from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours, from30-45 hours, from 30-40 hours, from 30-35 hours, from 40-94 hours, from40-90 hours, from 40-80 hours, from 40-70 hours, from 40-60, from 40-55hours, from 40-50 hours, from 40-45 hours, from 50-94 hours, from 50-90hours, from 50-80 hours, from 50-70 hours, from 50-60, from 60-94 hours,from 60-90 hours, from 60-80 hours, or such as a duration of from 60-70hours.

In specific embodiments, the incubation in step ii) of the process has aduration of from 2-35 hours and said incubation in step iii) of theprocess has a duration of from 2-94 hours, such as from 2-90 hours, from2-80 hours, from 2-70 hours, from 2-60, from 2-55 hours, from 2-50hours, from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours,from 2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours, from2-12 hours, from 2-10 hours, from 2-8 hours, from 4-94 hours, from 4-90hours, from 4-80 hours, from 4-70 hours, from 4-60, from 4-55 hours,from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35 hours, from4-30 hours, from 4-28 hours, from 4-26 hours, from 4-24, from 4-22hours, from 4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-94hours, from 10-90 hours, from 10-80 hours, from 10-70 hours, from 10-60,from 10-55 hours, from 10-50 hours, from 10-45 hours, from 10-40 hours,from 10-35 hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,from 10-24, from 10-22 hours, from 10-20 hours, from 10-18 hours, from10-16 hours, from 10-14 hours, from 10-12 hours, from 14-94 hours, from14-90 hours, from 14-80 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-94 hours, from 20-90 hours, from 20-80 hours, from 20-70 hours,from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours, from20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours, from20-26 hours, from 20-24, from 20-22 hours, from 24-94 hours, from 24-90hours, from 24-80 hours, from 24-70 hours, from 24-60, from 24-55 hours,from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35 hours,from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-94 hours,from 30-90 hours, from 30-80 hours, from 30-70 hours, from 30-60, from30-55 hours, from 30-50 hours, from 30-45 hours, from 30-40 hours, from30-35 hours, from 40-94 hours, from 40-90 hours, from 40-80 hours, from40-70 hours, from 40-60, from 40-55 hours, from 40-50 hours, from 40-45hours, from 50-94 hours, from 50-90 hours, from 50-80 hours, from 50-70hours, from 50-60, from 60-94 hours, from 60-90 hours, from 60-80 hours,or such as a duration of from 60-70 hours.

In other specific embodiments, the said incubation in step ii) of theprocess has a duration of from 2-20 hours and said incubation in stepiii) of the process has a duration of from 2-94 hours, such as from 2-90hours, from 2-80 hours, from 2-70 hours, from 2-60, from 2-55 hours,from 2-50 hours, from 2-45 hours, from 2-40 hours, from 2-35 hours, from2-30 hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22hours, from 2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14hours, from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-94hours, from 4-90 hours, from 4-80 hours, from 4-70 hours, from 4-60,from 4-55 hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours, from 4-16hours, from 4-14 hours, from 4-12 hours, from 4-10 hours, from 4-8hours, from 10-94 hours, from 10-90 hours, from 10-80 hours, from 10-70hours, from 10-60, from 10-55 hours, from 10-50 hours, from 10-45 hours,from 10-40 hours, from 10-35 hours, from 10-30 hours, from 10-28 hours,from 10-26 hours, from 10-24, from 10-22 hours, from 10-20 hours, from10-18 hours, from 10-16 hours, from 10-14 hours, from 10-12 hours, from14-94 hours, from 14-90 hours, from 14-80 hours, from 14-70 hours, from14-60, from 14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40hours, from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26hours, from 14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,from 14-16 hours, from 20-94 hours, from 20-90 hours, from 20-80 hours,from 20-70 hours, from 20-60, from 20-55 hours, from 20-50 hours, from20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30 hours, from20-28 hours, from 20-26 hours, from 20-24, from 20-22 hours, from 24-94hours, from 24-90 hours, from 24-80 hours, from 24-70 hours, from 24-60,from 24-55 hours, from 24-50 hours, from 24-45 hours, from 24-40 hours,from 24-35 hours, from 24-30 hours, from 24-28 hours, from 24-26 hours,from 30-94 hours, from 30-90 hours, from 30-80 hours, from 30-70 hours,from 30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours, from30-40 hours, from 30-35 hours, from 40-94 hours, from 40-90 hours, from40-80 hours, from 40-70 hours, from 40-60, from 40-55 hours, from 40-50hours, from 40-45 hours, from 50-94 hours, from 50-90 hours, from 50-80hours, from 50-70 hours, from 50-60, from 60-94 hours, from 60-90 hours,from 60-80 hours, or such as a duration of from 60-70 hours.

In still other embodiments, the said incubation in step ii) of theprocess has a duration of from 4-35 hours and said incubation in stepiii) of the process has a duration of from 2-92 hours, such as from 2-90hours, from 2-80 hours, from 2-70 hours, from 2-60, from 2-55 hours,from 2-50 hours, from 2-45 hours, from 2-40 hours, from 2-35 hours, from2-30 hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22hours, from 2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14hours, from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-92hours, from 4-90 hours, from 4-80 hours, from 4-70 hours, from 4-60,from 4-55 hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours, from 4-16hours, from 4-14 hours, from 4-12 hours, from 4-10 hours, from 4-8hours, from 10-92 hours, from 10-90 hours, from 10-80 hours, from 10-70hours, from 10-60, from 10-55 hours, from 10-50 hours, from 10-45 hours,from 10-40 hours, from 10-35 hours, from 10-30 hours, from 10-28 hours,from 10-26 hours, from 10-24, from 10-22 hours, from 10-20 hours, from10-18 hours, from 10-16 hours, from 10-14 hours, from 10-12 hours, from14-92 hours, from 14-90 hours, from 14-80 hours, from 14-70 hours, from14-60, from 14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40hours, from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26hours, from 14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,from 14-16 hours, from 20-92 hours, from 20-90 hours, from 20-80 hours,from 20-70 hours, from 20-60, from 20-55 hours, from 20-50 hours, from20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30 hours, from20-28 hours, from 20-26 hours, from 20-24, from 20-22 hours, from 24-92hours, from 24-90 hours, from 24-80 hours, from 24-70 hours, from 24-60,from 24-55 hours, from 24-50 hours, from 24-45 hours, from 24-40 hours,from 24-35 hours, from 24-30 hours, from 24-28 hours, from 24-26 hours,from 30-92 hours, from 30-90 hours, from 30-80 hours, from 30-70 hours,from 30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours, from30-40 hours, from 30-35 hours, from 40-92 hours, from 40-90 hours, from40-80 hours, from 40-70 hours, from 40-60, from 40-55 hours, from 40-50hours, from 40-45 hours, from 50-92 hours, from 50-90 hours, from 50-80hours, from 50-70 hours, from 50-60, from 60-94 hours, from 60-90 hours,from 60-80 hours, or such as a duration of from 60-70 hours.

In further embodiments, the said incubation in step ii) of the processhas a duration of from 4-22 hours, and said incubation in step iii) ofthe process has a duration of from 2-92 hours, such as from 2-90 hours,from 2-80 hours, from 2-70 hours, from 2-60, from 2-55 hours, from 2-50hours, from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours,from 2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours, from2-12 hours, from 2-10 hours, from 2-8 hours, from 4-92 hours, from 4-90hours, from 4-80 hours, from 4-70 hours, from 4-60, from 4-55 hours,from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35 hours, from4-30 hours, from 4-28 hours, from 4-26 hours, from 4-24, from 4-22hours, from 4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-92hours, from 10-90 hours, from 10-80 hours, from 10-70 hours, from 10-60,from 10-55 hours, from 10-50 hours, from 10-45 hours, from 10-40 hours,from 10-35 hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,from 10-24, from 10-22 hours, from 10-20 hours, from 10-18 hours, from10-16 hours, from 10-14 hours, from 10-12 hours, from 14-92 hours, from14-90 hours, from 14-80 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-92 hours, from 20-90 hours, from 20-80 hours, from 20-70 hours,from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours, from20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours, from20-26 hours, from 20-24, from 20-22 hours, from 24-92 hours, from 24-90hours, from 24-80 hours, from 24-70 hours, from 24-60, from 24-55 hours,from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35 hours,from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-92 hours,from 30-90 hours, from 30-80 hours, from 30-70 hours, from 30-60, from30-55 hours, from 30-50 hours, from 30-45 hours, from 30-40 hours, from30-35 hours, from 40-92 hours, from 40-90 hours, from 40-80 hours, from40-70 hours, from 40-60, from 40-55 hours, from 40-50 hours, from 40-45hours, from 50-92 hours, from 50-90 hours, from 50-80 hours, from 50-70hours, from 50-60, from 60-94 hours, from 60-90 hours, from 60-80 hours,or such as a duration of from 60-70 hours.

In still further embodiments, the said incubation in step ii) of theprocess has a duration of from 10-35 hours, and said incubation in stepiii) of the process has a duration of from 2-86 hours, such as from 2-80hours, from 2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours,from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from 2-20hours, from 2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12hours, from 2-10 hours, from 2-8 hours, from 4-86 hours, from 4-80hours, from 4-70 hours, from 4-60, from 4-55 hours, from 4-50 hours,from 4-45 hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from 4-20hours, from 4-18 hours, from 4-16 hours, from 4-14 hours, from 4-12hours, from 4-10 hours, from 4-8 hours, from 10-86 hours, from 10-80hours, from 10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,from 10-28 hours, from 10-26 hours, from 10-24, from 10-22 hours, from10-20 hours, from 10-18 hours, from 10-16 hours, from 10-14 hours, from10-12 hours, from 14-86 hours, from 14-80 hours, from 14-70 hours, from14-60, from 14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40hours, from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26hours, from 14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,from 14-16 hours, from 20-86 hours, from 20-80 hours, from 20-70 hours,from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours, from20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours, from20-26 hours, from 20-24, from 20-22 hours, from 25-86 hours, from 20-80hours, from 20-70 hours, from 20-60, from 20-55 hours, from 20-50 hours,from 20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30 hours,from 20-28 hours, from 20-26 hours, from 20-24, from 20-22 hours, from24-86 hours, from 24-80 hours, from 24-70 hours, from 24-60, from 24-55hours, from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35hours, from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-86hours, from 30-80 hours, from 30-70 hours, from 30-60, from 30-55 hours,from 30-50 hours, from 30-45 hours, from 30-40 hours, from 30-35 hours,from 40-86 hours, from 40-80 hours, from 40-70 hours, from 40-60, from40-55 hours, from 40-50 hours, from 40-45 hours, from 50-86 hours, from50-80 hours, from 50-70 hours, from 50-60, from 60-94 hours, from 60-90hours, from 60-80 hours, or such as a duration of from 60-70 hours.

In even further embodiments, the said incubation in step ii) of theprocess has a duration of from 10-24 hours, and said incubation in stepiii) of the process has a duration of from 2-86 hours, such as from 2-80hours, from 2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours,from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from 2-20hours, from 2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12hours, from 2-10 hours, from 2-8 hours, from 4-86 hours, from 4-80hours, from 4-70 hours, from 4-60, from 4-55 hours, from 4-50 hours,from 4-45 hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from 4-20hours, from 4-18 hours, from 4-16 hours, from 4-14 hours, from 4-12hours, from 4-10 hours, from 4-8 hours, from 10-86 hours, from 10-80hours, from 10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,from 10-28 hours, from 10-26 hours, from 10-24, from 10-22 hours, from10-20 hours, from 10-18 hours, from 10-16 hours, from 10-14 hours, from10-12 hours, from 14-86 hours, from 14-80 hours, from 14-70 hours, from14-60, from 14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40hours, from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26hours, from 14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,from 14-16 hours, from 20-86 hours, from 20-80 hours, from 20-70 hours,from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours, from20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours, from20-26 hours, from 20-24, from 20-22 hours, from 25-86 hours, from 20-80hours, from 20-70 hours, from 20-60, from 20-55 hours, from 20-50 hours,from 20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30 hours,from 20-28 hours, from 20-26 hours, from 20-24, from 20-22 hours, from24-86 hours, from 24-80 hours, from 24-70 hours, from 24-60, from 24-55hours, from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35hours, from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-86hours, from 30-80 hours, from 30-70 hours, from 30-60, from 30-55 hours,from 30-50 hours, from 30-45 hours, from 30-40 hours, from 30-35 hours,from 40-86 hours, from 40-80 hours, from 40-70 hours, from 40-60, from40-55 hours, from 40-50 hours, from 40-45 hours, from 50-86 hours, from50-80 hours, from 50-70 hours, from 50-60, from 60-94 hours, from 60-90hours, from 60-80 hours, or such as a duration of from 60-70 hours.

In yet further embodiments, the said incubation in step ii) of theprocess has a duration of from 14-35 hours, and said incubation in stepiii) of the process has a duration of from 2-70 hours, such as from2-60, from 2-55 hours, from 2-50 hours, from 2-45 hours, from 2-40hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from 2-26hours, from 2-24, from 2-22 hours, from 2-20 hours, from 2-18 hours,from 2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10 hours, from2-8 hours, from 4-70 hours, from 4-60, from 4-55 hours, from 4-50 hours,from 4-45 hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from 4-20hours, from 4-18 hours, from 4-16 hours, from 4-14 hours, from 4-12hours, from 4-10 hours, from 4-8 hours, from 10-70 hours, from 10-60,from 10-55 hours, from 10-50 hours, from 10-45 hours, from 10-40 hours,from 10-35 hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,from 10-24, from 10-22 hours, from 10-20 hours, from 10-18 hours, from10-16 hours, from 10-14 hours, from 10-12 hours, from 14-70 hours, from14-60, from 14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40hours, from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26hours, from 14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,from 14-16 hours, from 20-70 hours, from 20-60, from 20-55 hours, from20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours, from20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from 20-22hours, from 25-94 hours, from 25-90 hours, from 20-80 hours, from 20-70hours, from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours,from 20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,from 20-26 hours, from 20-24, from 20-22 hours, from 24-70 hours, from24-60, from 24-55 hours, from 24-50 hours, from 24-45 hours, from 24-40hours, from 24-35 hours, from 24-30 hours, from 24-28 hours, from 24-26hours, from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours,from 30-45 hours, from 30-40 hours, from 30-35 hours, from 40-70 hours,from 40-60, from 40-55 hours, from 40-50 hours, from 40-45 hours, from50-70 hours, from 50-60, from 60-94 hours, from 60-90 hours, from 60-80hours, or such as a duration of from 60-70 hours.

In even further embodiments, the incubation in step ii) of the processhas a duration of from 14-26 hours, and said incubation in step iii) ofthe process has a duration of from 2-70 hours, such as from 2-60, from2-55 hours, from 2-50 hours, from 2-45 hours, from 2-40 hours, from 2-35hours, from 2-30 hours, from 2-28 hours, from 2-26 hours, from 2-24,from 2-22 hours, from 2-20 hours, from 2-18 hours, from 2-16 hours, from2-14 hours, from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-70hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45 hours,from 4-40 hours, from 4-35 hours, from 4-30 hours, from 4-28 hours, from4-26 hours, from 4-24, from 4-22 hours, from 4-20 hours, from 4-18hours, from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10hours, from 4-8 hours, from 10-70 hours, from 10-60, from 10-55 hours,from 10-50 hours, from 10-45 hours, from 10-40 hours, from 10-35 hours,from 10-30 hours, from 10-28 hours, from 10-26 hours, from 10-24, from10-22 hours, from 10-20 hours, from 10-18 hours, from 10-16 hours, from10-14 hours, from 10-12 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-70 hours, from 20-60, from 20-55 hours, from 20-50 hours, from20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30 hours, from20-28 hours, from 20-26 hours, from 20-24, from 20-22 hours, from 25-94hours, from 25-90 hours, from 20-80 hours, from 20-70 hours, from 20-60,from 20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40 hours,from 20-35 hours, from 20-30 hours, from 20-28 hours, from 20-26 hours,from 20-24, from 20-22 hours, from 24-70 hours, from 24-60, from 24-55hours, from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35hours, from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-70hours, from 30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,from 30-40 hours, from 30-35 hours, from 40-70 hours, from 40-60, from40-55 hours, from 40-50 hours, from 40-45 hours, from 50-70 hours, from50-60, from 60-94 hours, from 60-90 hours, from 60-80 hours, or such asa duration of from 60-70 hours.

In other embodiments, the said incubation in step ii) of the process hasa duration of from 18-35 hours, and said incubation in step iii) of theprocess has a duration of from 2-78 hours, such as from 2-74 hours, from2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from 2-28hours, from 2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours,from 2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12 hours, from2-10 hours, from 2-8 hours, from 4-78 hours, from 4-74 hours, from 4-70hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45 hours,from 4-40 hours, from 4-35 hours, from 4-30 hours, from 4-28 hours, from4-26 hours, from 4-24, from 4-22 hours, from 4-20 hours, from 4-18hours, from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10hours, from 4-8 hours, from 10-76 hours, from 10-74 hours, from 10-70hours, from 10-60, from 10-55 hours, from 10-50 hours, from 10-45 hours,from 10-40 hours, from 10-35 hours, from 10-30 hours, from 10-28 hours,from 10-26 hours, from 10-24, from 10-22 hours, from 10-20 hours, from10-18 hours, from 10-16 hours, from 10-14 hours, from 10-12 hours, from14-78 hours, from 14-74 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-78 hours, from 20-74 hours, from 20-80 hours, from 20-70 hours,from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours, from20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours, from20-26 hours, from 20-24, from 20-22 hours, from 24-78 hours, from 24-76hours, from 24-70 hours, from 24-60, from 24-55 hours, from 24-50 hours,from 24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30 hours,from 24-28 hours, from 24-26 hours, from 30-78 hours, from 30-74 hours,from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours, from30-45 hours, from 30-40 hours, from 30-35 hours, from 40-76 hours, from40-74 hours, from 40-70 hours, from 40-60, from 40-55 hours, from 40-50hours, from 40-45 hours, from 50-78 hours, from 50-74 hours, from 50-70hours, from 50-60, from 60-94 hours, from 60-90 hours, from 60-80 hours,or such as a duration of from 60-70 hours.

In further embodiments, the said incubation in step ii) of the processhas a duration of from 18-28 hours, and said incubation in step iii) ofthe process has a duration of from 2-78 hours, such as from 2-74 hours,from 2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from 2-28hours, from 2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours,from 2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12 hours, from2-10 hours, from 2-8 hours, from 4-78 hours, from 4-74 hours, from 4-70hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45 hours,from 4-40 hours, from 4-35 hours, from 4-30 hours, from 4-28 hours, from4-26 hours, from 4-24, from 4-22 hours, from 4-20 hours, from 4-18hours, from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10hours, from 4-8 hours, from 10-76 hours, from 10-74 hours, from 10-70hours, from 10-60, from 10-55 hours, from 10-50 hours, from 10-45 hours,from 10-40 hours, from 10-35 hours, from 10-30 hours, from 10-28 hours,from 10-26 hours, from 10-24, from 10-22 hours, from 10-20 hours, from10-18 hours, from 10-16 hours, from 10-14 hours, from 10-12 hours, from14-78 hours, from 14-74 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-78 hours, from 20-74 hours, from 20-80 hours, from 20-70 hours,from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours, from20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours, from20-26 hours, from 20-24, from 20-22 hours, from 24-78 hours, from 24-76hours, from 24-70 hours, from 24-60, from 24-55 hours, from 24-50 hours,from 24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30 hours,from 24-28 hours, from 24-26 hours, from 30-78 hours, from 30-74 hours,from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours, from30-45 hours, from 30-40 hours, from 30-35 hours, from 40-76 hours, from40-74 hours, from 40-70 hours, from 40-60, from 40-55 hours, from 40-50hours, from 40-45 hours, from 50-78 hours, from 50-74 hours, from 50-70hours, from 50-60, from 60-94 hours, from 60-90 hours, from 60-80 hours,or such as a duration of from 60-70 hours.

In even further embodiments, the said incubation in step ii) of theprocess has a duration of from 20-35 hours, and said incubation in stepiii) of the process has a duration of from 2-76 hours, such as from 2-70hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45 hours,from 2-40 hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours, from 2-18hours, from 2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10hours, from 2-8 hours, from 4-76 hours, from 4-70 hours, from 4-60, from4-55 hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from 4-24,from 4-22 hours, from 4-20 hours, from 4-18 hours, from 4-16 hours, from4-14 hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-76hours, from 10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,from 10-28 hours, from 10-26 hours, from 10-24, from 10-22 hours, from10-20 hours, from 10-18 hours, from 10-16 hours, from 10-14 hours, from10-12 hours, from 14-76 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-76 hours, from 20-70 hours, from 20-60, from 20-55 hours, from20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours, from20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from 20-22hours, from 25-76 hours, from 20-70 hours, from 20-60, from 20-55 hours,from 20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours,from 20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from20-22 hours, from 24-76 hours, from 24-70 hours, from 24-60, from 24-55hours, from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35hours, from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-76hours, from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours,from 30-45 hours, from 30-40 hours, from 30-35 hours, from 40-76 hours,from 40-70 hours, from 40-60, from 40-55 hours, from 40-50 hours, from40-45 hours, from 50-76 hours, from 50-70 hours, from 50-60, from 60-94hours, from 60-90 hours, from 60-80 hours, or such as a duration of from60-70 hours.

In still further embodiments, the said incubation in step ii) of theprocess has a duration of from 20-30 hours, and said incubation in stepiii) of the process has a duration of from 2-76 hours, such as from 2-70hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45 hours,from 2-40 hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours, from 2-18hours, from 2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10hours, from 2-8 hours, from 4-76 hours, from 4-70 hours, from 4-60, from4-55 hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from 4-24,from 4-22 hours, from 4-20 hours, from 4-18 hours, from 4-16 hours, from4-14 hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-76hours, from 10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,from 10-28 hours, from 10-26 hours, from 10-24, from 10-22 hours, from10-20 hours, from 10-18 hours, from 10-16 hours, from 10-14 hours, from10-12 hours, from 14-76 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-76 hours, from 20-70 hours, from 20-60, from 20-55 hours, from20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours, from20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from 20-22hours, from 25-76 hours, from 20-70 hours, from 20-60, from 20-55 hours,from 20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours,from 20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from20-22 hours, from 24-76 hours, from 24-70 hours, from 24-60, from 24-55hours, from 24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35hours, from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-76hours, from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours,from 30-45 hours, from 30-40 hours, from 30-35 hours, from 40-76 hours,from 40-70 hours, from 40-60, from 40-55 hours, from 40-50 hours, from40-45 hours, from 50-76 hours, from 50-70 hours, from 50-60, from 60-94hours, from 60-90 hours, from 60-80 hours, or such as a duration of from60-70 hours.

In even further embodiments, the said incubation in step ii) of theprocess has a duration of from 22-35 hours, and said incubation in stepiii) of the process has a duration of from 2-74 hours, such as from 2-70hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45 hours,from 2-40 hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours, from 2-18hours, from 2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10hours, from 2-8 hours, from 4-74 hours, from 4-70 hours, from 4-60, from4-55 hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from 4-24,from 4-22 hours, from 4-20 hours, from 4-18 hours, from 4-16 hours, from4-14 hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-74hours, from 10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,from 10-28 hours, from 10-26 hours, from 10-24, from 10-22 hours, from10-20 hours, from 10-18 hours, from 10-16 hours, from 10-14 hours, from10-12 hours, from 14-74 hours, from 14-70 hours, from 14-60, from 14-55hours, from 14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35hours, from 14-30 hours, from 14-28 hours, from 14-26 hours, from 14-24,from 14-22 hours, from 14-20 hours, from 14-18 hours, from 14-16 hours,from 20-74 hours, from 20-70 hours, from 20-60, from 20-55 hours, from20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35 hours, from20-30 hours, from 20-28 hours, from 20-26 hours, from 20-24, from 20-22hours, from 25-94 hours, from 25-90 hours, from 20-80 hours, from 20-70hours, from 20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours,from 20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,from 20-26 hours, from 20-24, from 20-22 hours, from 24-74 hours, from24-70 hours, from 24-60, from 24-55 hours, from 24-50 hours, from 24-45hours, from 24-40 hours, from 24-35 hours, from 24-30 hours, from 24-28hours, from 24-26 hours, from 30-94 hours, from 30-90 hours, from 30-80hours, from 30-70 hours, from 30-60, from 30-55 hours, from 30-50 hours,from 30-45 hours, from 30-40 hours, from 30-35 hours, from 40-74 hours,from 40-70 hours, from 40-60, from 40-55 hours, from 40-50 hours, from40-45 hours, from 50-94 hours, from 50-90 hours, from 50-80 hours, from50-70 hours, from 50-60, from 60-74 hours, or such as a duration of from60-70 hours.

The incubation in step iii) of claim 14 may be allowed to proceed untilthe amount of glucose (% DX) in the composition has reached a levelcorresponding to 95% (w/w) or more, such as 95.5% (w/w) or more, 95.75%(w/w) or more, 96% (w/w) or more, 96.25% (w/w) or more, or such as96.75% (w/w) or more.

In some embodiments, the duration of the process from addition of said aglucoamylase (AMG) and said pullulanase to the composition comprisingliquefied starch until termination of the starchhydrolysis/saccharification is from 24-96 hours, such as from 24-72hours, from 24-64 hours, from 24-48 hours, from 24-36 hours, from 24-32hours, from 32-96 hours, from 32-72 hours, from 32-64 hours, from 32-48hours, from 32-36 hours, from 36-96 hours, from 36-72 hours, from 36-64hours, from 36-48 hours, from 48-96 hours, such as from 48-72 hours,from 48-64 hours, from 56-96 hours, such as from 56-72 hours, or such asfrom 56-64 hours,

In the process according to the invention, the starchhydrolysis/saccharification may in particular take place at a pH whichis within the range of 3.5-5.0, such as at pH in the range of 4.0-4.5,and at a temperature, which is within the range of 59-70° C., such as inthe range of 59-65° C. or such as in the range of 59-62° C.

The liquefied starch used as substrate for the saccharification processaccording to the invention may be a starch slurry or partiallyhydrolysed starch (liquefact or maltodextrin). In particular, the instarch slurry or partly hydrolysed starch may have a Dextrose equivalent(DE) in the range of 5-42, such as in the range of 5-30, in the range of8-18 or such as in the range of 9-14.

The starch may be from any source, in particular from corn, wheat ortapioca. The starch slurry or partially hydrolysed starch may haveresidual alpha amylase activity from the liquefaction process present orit may have been deactivated, such as by reducing the pH to below 4.5,while maintaining a high temperature (above 95° C.), to inactivate theliquefying alpha-amylase.

The conductivity of said starch slurry or partially hydrolysed starchmay in particular be within the range of 0-500 microS/cm. According tosome embodiments the calcium content corresponds to 0-200 ppm freecalcium.

In the process according to the invention, the starchhydrolysis/saccharification may in particular take place at a pH whichis within the range of 3.5-5.0, such as at pH in the range of 4.0-4.7,and at a temperature, which is within the range of 58−70° C., such as inthe range of 58-65° C., in the range of 59−65° C. or such as in therange of 59−62° C.

The composition comprising liquefied starch provided as a startingmaterial; i.e. composition comprising liquefied starch provided the instep i) of the process may contain from 25-45% dry solids (% DS), suchas from 25-40% DS, from 30-38% DS, from 32-38% DS, or such as from34-36% DS.

Fungal alpha-amylases useful in the processes according to the inventioninclude alpha-amylases derived from a strain of the genus Aspergillus,such as, Aspergillus oryzae, Aspergillus niger and Aspergillus kawachiialpha-amylases.

A preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is derived from a strain of Aspergillus oryzae. According to thepresent invention, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e., more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acidic alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in WO 89/01969 (Example 3).

Other contemplated wild-type alpha-amylases include those derived from astrain of the genera Rhizomucor and Meripilus, preferably a strain ofRhizomucor pusillus (WO 2004/055178 incorporated by reference) orMeripilus giganteus.

In a preferred embodiment, the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81:292-298, “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii”; and further as EMBL:#AB008370.

The fungal alpha-amylase may also be a wild-type enzyme comprising astarch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e.,non-hybrid), or a variant thereof. In an embodiment the wild-typealpha-amylase is derived from a strain of Aspergillus kawachii.

In a further embodiment, the alpha amylase is a fungal acidalpha-amylase is a hybrid alpha-amylase. Preferred examples of fungalhybrid alpha-amylases include the ones disclosed in WO 2005/003311 orU.S. application publication no. 2005/0054071 (Novozymes) or U.S.application No. 60/638,614 (Novozymes) which is hereby incorporated byreference. A hybrid alpha-amylase may comprise an alpha-amylasecatalytic domain (CD) and a carbohydratebinding domain/module (CBM),such as a starch binding domain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in Table 1 to 5 of the examples in U.S. application No.60/638,614, including Fungamyl variant with catalytic domain JA118 andAthelia rolfsii SBD (SEQ ID NO:100 in U.S. 60/638,614), Rhizomucorpusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ IDNO:101 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase withAspergillus niger glucoamylase linker and SBD (which is disclosed inTable 5 as a combination of amino acid sequences SEQ ID NO: 20, SEQ IDNO: 72 and SEQ ID NO: 96 in U.S. application Ser. No. 11/316,535) or asV039 in Table 5 in WO 2006/069290, and Meripilus giganteus alpha-amylasewith Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S.application No. 60/638,614). Other specifically contemplated hybridalpha-amylases are any of the ones listed in Tables 3, 4, 5, and 6 inExample 4 in U.S. application Ser. No. 11/316,535 and WO 2006/069290(hereby incorporated by reference).

Other specific examples of contemplated hybrid alpha-amylases includethose disclosed in U.S. application publication no. 2005/0054071,including those disclosed in Table 3 on page 15, such as Aspergillusniger alpha-amylase with Aspergillus kawachii linker and starch bindingdomain.

Bacterial alpha-amylases useful in the processes according to theinvention include alpha-amylases derived from a strain of the genusBacillus, such as Bacillus licheniformis, Bacillus stearothermophilus.

Contemplated are also alpha-amylases which exhibit a high identity toany of the above mentioned alpha-amylases, i.e., more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature enzyme sequences.

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE™ (DSM), BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X, LIQUOZYME™SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes NS) and CLARASE™ L-40,000,DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ ALPHA, SPEZYME™ DELTA AA,GC358, GC980, SPEZYME™ CL and SPEZYME™ RSL (DuPont-Genencor), FUELZYME™(from Verenium Corp, USA).

These alpha amylases may, if desired, also be included in thecompositions according to the invention at the very low amounts setforth above.

Glucoamylases and pullulanases which are useful in the processesaccording to the present invention include those disclosed above inrelation to the compositions according to the invention.

Hence, in particular embodiments of the invention the alpha amylase isselected from Aspergillus niger, Aspergillus terreus or Rhizomucorpusillus alpha amylases, the glucoamylase is selected from Aspergillusniger, Aspergillus fumigatus, Talaromyces emersonii, Trametes cingulata,Trichoderma reesei, Humicloa insolens and Gloeophyllum trabeumglucoamylases, and the pullulanase is selected from Bacillusderamificans or Bacillus acidopullulyticus pullulanases.

Specifically, the glucoamylase may comprise an amino acid sequenceselected from the group consisting of

-   -   i) The amino acid sequence set forth in any of SEQ ID NOs: 2-12        and 27 or a mature polypeptide thereof;    -   ii) A subsequence of the amino acid sequence set forth in any        one of SEQ ID NOs: 2-12 and 27 or of said mature polypeptide        thereof;    -   iii) A variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in i) and ii).

In related embodiments, the glucoamylase consists essentially of anamino acid sequence as set forth in any of items i)-iii) above. In otherrelated embodiments the glucoamylase consists of an amino acid sequenceas set forth in any of items i)-iii) above.

The alpha-amylase used in the process according to the inventioncomprises/consists essentially of/consists of an amino acid sequenceselected from the group consisting of

-   -   i) The amino acid sequence set forth in any one of SEQ ID NOs: 1        and 17-26 or a mature polypeptide thereof;    -   ii) A subsequence of the amino acid sequence set forth in any        one of SEQ ID NOs: 1 and 17-26 or of said mature polypeptide        thereof;    -   iii) A variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in i) and ii).

In related embodiments, the alpha-amylase consists essentially of anamino acid sequence as set forth in any of items i)-iii) above. In otherrelated embodiments the alpha-amylase consists of an amino acid sequenceas set forth in any of items i)-iii) above.

In particular embodiments, the alpha-amylase comprising or consisting ofthe amino acid sequence defined in iii) is a variant of an alpha-amylasecomprising or consisting of the amino sequence defined in SEQ ID NO: 20or a mature polypeptide thereof, wherein the following mutations havebeen made: 1181*/G182*/N193F (using the amino acid numbering in SEQ IDNO: 20).

According to other embodiments, the alpha-amylase comprising orconsisting of the amino acid sequence defined in iii) is a variant of analpha-amylase comprising or consisting of the amino sequence defined inSEQ ID NO: 23 or a mature polypeptide thereof, wherein the followingmutations have been made: H156Y+A181T+N190F+A209V+Q264S (using the aminoacid numbering in SEQ ID NO: 21).

In even further embodiments, the alpha-amylase comprising or consistingof the amino acid sequence defined in iii) is a variant of analpha-amylase comprising or consisting of the amino sequence defined inSEQ ID NO: 23 or a mature polypeptide thereof, wherein the followingmutations have been made:G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the numberingin SEQ ID NO: 21).

The pullulanase used in the process according to the invention maycomprises/consists essentially of/consists of an amino acid sequenceselected from the group consisting of

-   -   i) The amino acid sequence set forth in any of SEQ ID NOs: 13-16        or a mature polypeptide thereof;    -   ii) A subsequence of the amino acid sequence set forth in any        one of SEQ ID NOs: 13-16 or of said mature polypeptide thereof;    -   iii) A variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in i) and ii).

In related embodiments, the pullulanase consists essentially of an aminoacid sequence as set forth in any of items i)-iii) above. In otherrelated embodiments the pullulanase consists of an amino acid sequenceas set forth in any of items i)-iii) above.

Within the scope of the present invention are embodiments wherein theglucoamylase comprises or consists of an amino acid sequence selectedfrom the group consisting of:

-   -   i) the amino acid sequence set forth in SEQ ID NO: 12 or a        mature polypeptide thereof,    -   ii) a subsequence of the amino acid sequence set forth in SEQ ID        NO: 12 or of said mature polypeptide thereof, and    -   iii) a variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in i) and ii);

wherein the pullulanase comprises or consists of an amino acid sequenceselected from the group consisting of:

-   -   iv) the amino acid sequence set forth in any one of SEQ ID NOs:        13-16 or a mature polypeptide thereof;    -   v) a subsequence of the amino acid sequence set forth in any one        of SEQ ID NOs: 13-16 or of said mature polypeptide thereof, and    -   vi) a variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in iv) and v); and

wherein the alpha-amylase comprises or consists of an amino acidsequence selected from the group consisting of:

-   -   vii) the amino acid sequence set forth in SEQ ID NO: 18 or a        mature polypeptide thereof,    -   viii) a subsequence of the amino acid sequence set forth in SEQ        ID NO: 18 or of said mature polypeptide thereof, and    -   iv) a variant amino acid sequence, which has at least 70%, such        as at least 75%, at least 80%, at least 85%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%,        such as at least 99.5% sequence identity to any one of the amino        acids sequences set forth in vii) and viii).

When said glucoamylase is a subsequence or a variant amino acid sequenceas defined above, it preferably has at least 80%, such as at least 90%of the glucoamylase activity of the respective amino acid of which it isa subsequence or variant, when tested as set forth above in relation tothe definition of “Glucoamylase activity (AGU)”.

When said pullulanase is a subsequence or a variant amino acid sequenceas defined above, it preferably has at least 80%, such as at least 90%of the pullulanase activity of the respective amino acid of which it isa subsequence or variant, when tested as set forth above in relation tothe definition of “Pullulanase activity (NPUN)”.

When the alpha-amylase defined above is a subsequence or a variant, itpreferably has at least 80%, such as at least 90% of the alpha-amylaseactivity of the respective alpha-amylase selected from SEQ ID NOs: 1 and17-26 or of the mature polypeptide thereof, of which it is a subsequenceor variant, when tested as set forth above in relation to the definitionof “Acid alpha-Amylase Units (FAU(A))” or “Alpha-amylase Activity(KNU)”: In the present context an alpha-amylase comprising an amino acidsequences selected from the group consisting of SEQ ID NOs: 1, 17-19 and26 or of a mature polypeptide thereof is considered to be a fungal alphaamylase and activity is tested as provided in relation to the abovedefinition of “Acid alphaAmylase Units (FAU(A))”. An alpha-amylasecomprising of an amino acid sequences selected from the group consistingof SEQ ID NOs: 20-25 or of a mature polypeptide thereof, is consideredto be a bacterial alpha amylase and activity is tested as provided inrelation to the above definition of “Alpha-amylase Activity (KNU)”.

In a further aspect, the invention provides a process for producingglucose syrup from liquefied starch. The process comprises contactingthe liquefied starch with a composition according to according to theinvention a described hereinabove.

The liquefied starch may in particular contain from 25-45% dry solids (%DS) (w/w %), such as from 25-40% DS (w/w %), from 30-38% DS, from 32-38%DS, or such as from 34-36% DS.

In further embodiments, the process comprises subjecting the liquifiedstarch to incubation under conditions allowing starchhydrolysis/saccharification.

Items

-   -   1. A composition comprising a glucoamylase and a pullulanase,        wherein the ratio of pullulanase activity expressed as NPUN/g to        glucoamylase activity expressed as AGU/g is higher than 6, such        as higher than 7, higher than 8, higher than 9 or within the        range of 6-20, such as within the range of 6-19, within the        range of 6-18. within the range of 6-17, within the range of        6-16, within the range of 6-15, within the range of 6-14, within        the range of 6-13, within the range of 6-12, within the range of        6-11, within the range of 7-20, within the range of 7-19, within        the range of 7-18, within the range of 7-17, within the range of        7-16, within the range of 7-15, within the range of 7-14, within        the range of 7-13, within the range of 7-12, within the range of        7-11, within the range of 8-20, within the range of 8-19, within        the range of 8-18, within the range of 8-17, within the range of        8-16, within the range of 8-15, within the range of 8-14, within        the range of 8-13, within the range of 8-12, within the range of        8-11, within the range of 9-20, within the range of 9-19, within        the range of 9-18, within the range of 9-17, within the range of        9-16, within the range of 9-15, within the range of 9-14, within        the range of 9-13, within the range of 9-12 or such as within        the range of 9-11.    -   2. The composition according to item 1, wherein if said        composition comprises a polypeptide having alpha-amylase        activity and that polypeptide is of fungal origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Fungal alpha-Amylase Units        FAU(A)/g is higher than 29, such as higher than 50, higher than        100, higher than 200, higher than 300, higher than 400, higher        than 500, higher than 600, higher than 800, higher than 1000,        higher than 2000, higher than 3000, higher than 4000 or such as        higher than 5000, and wherein if the polypeptide having        alpha-amylase activity is of bacterial origin, then the ratio of        glucoamylase activity expressed as AGU/g to alpha-amylase        activity expressed as Kilo Novo alpha-amylase Units (KNU)/g is        higher than 29, such as higher than 50, higher than 100, higher        than 200, higher than 300, higher than 400, higher than 500,        higher than 600, higher than 800, higher than 1000, higher than        2000, higher than 3000, higher than 4000 or such as higher than        5000.    -   3. The composition according to item 1 or 2, wherein the ratio        of pullulanase activity expressed as NPUN/g to glucoamylase        activity expressed as AGU/g is higher than 6, and wherein if        said composition comprises a polypeptide having alpha-amylase        activity and that polypeptide is of fungal origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Fungal alpha-Amylase Units        FAU(A)/g is higher than 100, and wherein if the polypeptide        having alpha-amylase activity is of bacterial origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Kilo Novo alpha-amylase        Units (KNU)/g is higher than 100.    -   4. The composition according to item 1 or 2, wherein the ratio        of pullulanase activity expressed as NPUN/g to glucoamylase        activity expressed as AGU/g is within the range of 6-20, and        wherein if said composition comprises a polypeptide having        alpha-amylase activity and that polypeptide is of fungal origin,        then the ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Fungal alpha-Amylase Units        FAU(A)/g is higher than 100, and wherein if the polypeptide        having alpha-amylase activity is of bacterial origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Kilo Novo alpha-amylase        Units (KNU)/g is higher than 100.    -   5. The composition according to item 1 or 2, wherein the ratio        of pullulanase activity expressed as NPUN/g to glucoamylase        activity expressed as AGU/g is within the range of 6-20, and        wherein if said composition comprises a polypeptide having        alpha-amylase activity and that polypeptide is of fungal origin,        then the ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Fungal alpha-Amylase Units        FAU(A)/g is higher than 500, and wherein if the polypeptide        having alpha-amylase activity is of bacterial origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Kilo Novo alpha-amylase        Units (KNU)/g is higher than 500    -   6. The composition according to item 1 or 2, wherein the ratio        of pullulanase activity expressed as NPUN/g to glucoamylase        activity expressed as AGU/g is within the range of 7-15, and        wherein if said composition comprises a polypeptide having        alpha-amylase activity and that polypeptide is of fungal origin,        then the ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Fungal alpha-Amylase Units        FAU(A)/g is higher than 100, and wherein if the polypeptide        having alpha-amylase activity is of bacterial origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Kilo Novo alpha-amylase        Units (KNU)/g is higher than 100.    -   7. The composition according to item 1 or 2, wherein the ratio        of pullulanase activity expressed as NPUN/g to glucoamylase        activity expressed as AGU/g is within the range of 7-15, and        wherein if said composition comprises a polypeptide having        alpha-amylase activity and that polypeptide is of fungal origin,        then the ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Fungal alpha-Amylase Units        FAU(A)/g is higher than 500, and wherein if the polypeptide        having alpha-amylase activity is of bacterial origin, then the        ratio of glucoamylase activity expressed as AGU/g to        alpha-amylase activity expressed as Kilo Novo alpha-amylase        Units (KNU)/g is higher than 100.    -   8. The composition according to any of the preceding items,        wherein said glucoamylase is selected from the group consisting        of an Aspergillus niger glucoamylase, a Talaromyces emersonii        glucoamylase, a Trametes cingulata glucoamylase a Gloeophyllum        trabeum glucoamylase, a Trichoderma Reesei glucoamylase, a        Humicola insolens glucoamylase and an Aspergillus fumigatus        glucoamylase, and hybrids and variants thereof.    -   9. The composition according to any of the preceding items,        wherein said pullulanase is selected from the group consisting        of a Bacillus deramificans pullulanase, a Bacillus        acidopullulyticus pullulanase, and hybrids and variants thereof.    -   10. The composition according to any of the preceding items,        wherein the glucoamylase comprises/consists essentially        of/consists of an amino acid sequence selected from the group        consisting of        -   i) The amino acid sequence set forth in any of SEQ ID NO:            2-12 and 27;        -   ii) A subsequence of the amino acid sequence set forth in            any one of SEQ ID NOs: 2-12 and 27;        -   iii) An amino acid sequence, which has at least 70%, such as            at least 75%, at least 80%, at least 85%, at least 90%, at            least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 96%, at least 97%, at least 98%, at            least 99%, such as at least 99.5% sequence identity to any            one of the amino acids sequences set forth in i) and ii).    -   11. The composition according to any of the preceding items,        wherein the alpha-amylase comprises/consists essentially        of/consists of an amino acid sequence selected from the group        consisting of        -   i) The amino acid sequence set forth in any of SEQ ID NOs: 1            and 17-26;        -   ii) A subsequence of the amino acid sequence set forth in            any one of SEQ ID NOs: 1 and 17-26;        -   iii) An amino acid sequence, which has at least 70%, such as            at least 75%, at least 80%, at least 85%, at least 90%, at            least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 96%, at least 97%, at least 98%, at            least 99%, such as at least 99.5% sequence identity to any            one of the amino acids sequences set forth in i) and ii).    -   12. The composition according to any of the preceding items,        wherein the pullulanase comprises/consists essentially        of/consists of an amino acid sequence selected from the group        consisting of        -   i) The amino acid sequence set forth in any one of SEQ ID            NOs: 13-16;        -   ii) A subsequence of the amino acid sequence set forth in            any one of SEQ ID NOs: 13-16;        -   iii) An amino acid sequence, which has at least 70%, such as            at least 75%, at least 80%, at least 85%, at least 90%, at            least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 96%, at least 97%, at least 98%, at            least 99%, such as at least 99.5% sequence identity to any            one of the amino acids sequences set forth in i) and ii).    -   13. The composition according to any of the preceding items,        said composition being a liquid or solid composition.    -   14. A process for producing glucose syrup comprising,        -   i) providing a composition comprising liquefied starch, and            adding to said composition a glucoamylase (AMG) and a            pullulanase;        -   ii) subjecting the composition to incubation under            conditions allowing starch hydrolysis/saccharification, and            subsequently        -   iii) adding an alpha-amylase to the said composition and            subjecting the composition to incubation under conditions            allowing starch hydrolysis/saccharification.    -   15. The process according to item 14, wherein the amounts of        glucoamylase added in step i) of item 14 correspond to 0.05-0.5        AGU/gDS, such as 0.05-0.4 AGU/gDS, 0.05-0.35 AGU/gDS, 0.05-0.3        AGU/gDS, 0.075-0.5 AGU/gDS, 0.075-0.4 AGU/gDS, 0.075-0.35        AGU/gDS 0.075-0.3 AGU/gDS, 0.075-0.5 AGU/gDS 0.1-0.5 AGU/gDS,        0.1-0.4 AGU/gDS, 0.1-0.35 AGU/gDS, 0.1-0.3 AGU/gDS, 0.125-0.5        AGU/gDS, 0.125-0.4 AGU/gDS, 0.125-0.35 AGU/gDS, 0.125-0.3        AGU/gDS, 0.15-0.5 AGU/gDS, 0.15-0.4 AGU/gDS, 0.15-0.35 AGU/gDS,        0.15-0.3 AGU/gDS, 0.175-0.5 AGU/gDS, 0.175-0.4 AGU/gDS,        0.175-0.35 AGU/gDS, 0.175-0.3 AGU/gDS, 0.2-0.5 AGU/gDS, 0.2-0.4        AGU/gDS, 0.2-0.35 AGU/gDS, 0.2-0.3 AGU/gDS, 0.24-0.5 AGU/gDS,        0.24-0.4 AGU/gDS, 0.24-0.35 AGU/gDS or such as 0.24-0.26        AGU/gDS.    -   16. The process according to any of items 14-15, wherein the        amounts of pullulanase added in step i) of item 14 correspond to        0.05-5 NPUN(X)/gDS, such as 0.05-4.5 NPUN(X)/gDS, 0.05-4        NPUN(X)/gDS, 0.05-3.5 NPUN(X)/gDS, 0.05-3 NPUN(X)/gDS, 0.05-2.5        NPUN(X)/gDS, 0.1-5 NPUN(X)/gDS, 0.1-4 NPUN(X)/gDS, 0.1-3.5        NPUN(X)/gDS, 0.1-3 NPUN(X)/gDS, 0.1-2.5 NPUN(X)/gDS, 0.25-5        NPUN(X)/gDS, 0.25-4.5 NPUN(X)/gDS, 0.25-4 NPUN(X)/gDS, 0.25-3.5        NPUN(X)/gDS, 0.25-3 NPUN(X)/gDS, 0.5-5 NPUN(X)/gDS, 0.5-4.5        NPUN(X)/gDS, 0.5-4 NPUN(X)/gDS, 0.5-3.5 NPUN(X)/gDS, 0.05-3        NPUN(X)/gDS, 0.5-2.5 NPUN(X)/gDS, 1.0-5 NPUN(X)/gDS, 1.0-4.5        NPUN(X)/gDS, 1.0-4 NPUN(X)/gDS, 1.0-3.5 NPUN(X)/gDS, 1.0-3        NPUN(X)/gDS, 1.0-2.5 NPUN(X)/gDS, 1.5-5 NPUN(X)/gDS, 1.5-4.5        NPUN(X)/gDS, 1.5-4 NPUN(X)/gDS, 1.5-3.5 NPUN(X)/gDS, 1.5-3        NPUN(X)/gDS, 1.5-2.5 NPUN(X)/gDS, 2-5 NPUN(X)/gDS, 2-4.5        NPUN(X)/gDS, 2-4 NPUN(X)/gDS, 2-3.5 NPUN(X)/gDS, 2-3        NPUN(X)/gDS, 2-5 NPUN(X)/gDS, 2-2.5 NPUN(X)/gDS, 2.25-5        NPUN(X)/gDS, 2.25-4.5 NPUN(X)/gDS, 2.25-4 NPUN(X)/gDS, 2.25-3.5        NPUN(X)/gDS, 2.25-3 NPUN(X)/gDS, 2.25-2.5 NPUN(X)/gDS, or such        as 2.50-2.54 NPUN(X)/gDS.    -   17. The process according to any of items 14-16, wherein the        amounts of alpha-amylase added in step iii) of item 14        correspond to 0.0005-0.025 (FAU)(A)/gDS, such as 0.0005-0.0225        (FAU)(A)/gDS, 0.0005-0.02 (FAU)(A)/gDS, 0.0005-0.0175        (FAU)(A)/gDS, 0.0005-0.015 (FAU)(A)/gDS, 0.0005-0.0125        (FAU)(A)/gDS, 0.001-0.025 (FAU)(A)/gDS, 0.001-0.0225        (FAU)(A)/gDS, 0.001-0.02 (FAU)(A)/gDS, 0.001-0.0175        (FAU)(A)/gDS, 0.001-0.015 (FAU)(A)/gDS, 0.001-0.0125        (FAU)(A)/gDS, 0.0025-0.025 (FAU)(A)/gDS, 0.0025-0.0225        (FAU)(A)/gDS, 0.0025-0.02 (FAU)(A)/gDS, 0.0025-0.0175        (FAU)(A)/gDS, 0.0025-0.015 (FAU)(A)/gDS, 0.0025-0.0125        (FAU)(A)/gDS, 0.005-0.025 (FAU)(A)/gDS, 0.005-0.0225        (FAU)(A)/gDS, 0.005-0.02 (FAU)(A)/gDS, 0.005-0.0175        (FAU)(A)/gDS, 0.005-0.015 (FAU)(A)/gDS, 0.005-0.0125        (FAU)(A)/gDS, 0.0075-0.025 (FAU)(A)/gDS, 0.0075-0.0225        (FAU)(A)/gDS, 0.0075-0.02 FAU(A)/gDS, 0.0075-0.0175 FAU(A)/gDS,        0.0075-0.015 FAU(A)/gDS, 0.0075-0.0125 FAU(A)/gDS, 0.008-0.025        (FAU)(A)/gDS, 0.008-0.0225 (FAU)(A)/gDS, 0.008-0.02        (FAU)(A)/gDS, 0.008-0.0175 (FAU)(A)/gDS, 0.008-0.015        (FAU)(A)/gDS, 0.008-0.0125 (FAU)(A)/gDS, 0.009-0.0125        (FAU)(A)/gDS or such as 0.01 FAU(A)/gDS, and the amounts of        alpha-amylase added in step iii) of item 14 correspond to        0.0005-0.025 KNU/gDS, such as 0.0005-0.0225 KNU/gDS, 0.0005-0.02        KNU/gDS, 0.0005-0.0175 KNU/gDS, 0.0005-0.015 KNU/gDS,        0.0005-0.0125 KNU/gDS, 0.001-0.025 KNU/gDS, 0.001-0.0225        KNU/gDS, 0.001-0.02 KNU/gDS, 0.001-0.0175 KNU/gDS, 0.001-0.015        KNU/gDS, 0.001-0.0125 KNU/gDS, 0.0025-0.025 KNU/gDS,        0.0025-0.0225 KNU/gDS, 0.0025-0.02 KNU/gDS, 0.0025-0.0175        KNU/gDS, 0.0025-0.015 KNU/gDS, 0.0025-0.0125 KNU/gDS,        0.005-0.025 KNU/gDS, 0.005-0.0225 KNU/gDS, 0.005-0.02 KNU/gDS,        0.005-0.0175 KNU/gDS, 0.005-0.015 KNU/gDS, 0.005-0.0125 KNU/gDS,        0.0075-0.025 KNU/gDS, 0.0075-0.0225 KNU/gDS, 0.0075-0.02        KNU/gDS, 0.0075-0.0175 KNU/gDS, 0.0075-0.015 KNU/gDS,        0.0075-0.0125 KNU/gDS, 0.008-0.025 KNU/gDS, 0.008-0.0225        KNU/gDS, 0.008-0.02 KNU/gDS, 0.008-0.0175 KNU/gDS, 0.008-0.015        KNU/gDS, 0.008-0.0125 KNU/gDS, 0.009-0.01 KNU/gDS or such as        0.0095 KNU/gDS. if said alpha-amylase is a bacterial        alpha-amylase.    -   18. The process according to any of items 14-17, wherein the        amounts of glucoamylase added in step i) of item 14 correspond        to 0.05-0.5 AGU/gDS, the amounts of pullulanase added in step i)        of item 14 correspond to 0.05-5 NPUN(X)/gDS, and the amounts of        alpha-amylase added in step iii) of item 14 correspond to        0.0005-0.025 (FAU)(A)/gDS if the alpha-amylase is a fungal        alpha-amylase and to 0.0005-0.025 KNU/gDS if said alpha-amylase        is a bacterial alpha-amylase.    -   19. The process according to any of items 14-17, wherein the        amounts of glucoamylase added in step i) of item 14 correspond        to 0.125-0.35 AGU/gDS, the amounts of pullulanase added in        step i) of item 14 correspond to 1.5-3.5 NPUN(X)/gDS, and the        amounts of alpha-amylase added in step iii) of item 14        correspond to 0.0025-0.02 (FAU)(A)/gDS if the alpha-amylase is a        fungal alpha-amylase and to 0.0025-0.02 KNU/gDS if said        alpha-amylase is a bacterial alpha-amylase.    -   20. The process according to any of items 14-17, wherein the        amounts of glucoamylase added in step i) of item 14 correspond        to 0.175-0.3 AGU/gDS, the amounts of pullulanase added in        step i) of item 14 correspond to 2-4 NPUN(X)/gDS, and the        amounts of alpha-amylase added in step iii) of item 14        correspond to 0.0075-0.0175 FAU(A)/gDS if the alpha-amylase is a        fungal alpha-amylase and to 0.0075-0.0175 KNU/gDS if said        alpha-amylase is a bacterial alpha-amylase.    -   21. The process according to any of items 14-17, wherein the        amounts of glucoamylase added in step i) of item 14 correspond        to 0.2-0.3 AGU/gDS, the amounts of pullulanase added in step i)        of item 14 correspond to 2-3 NPUN(X)/gDS, and the amounts of        alpha-amylase added in step iii) of item 14 correspond to        0.008-0.0125 (FAU)(A)/gDS if the alpha-amylase is a fungal        alpha-amylase and to 0.008-0.0125 KNU/gDS if said alpha-amylase        is a bacterial alpha-amylase.    -   22. The process according to any of items 14-21, comprising        adding to said composition comprising liquefied starch a        formulation comprising a glucoamylase and a pullulanase, wherein        if said formulation comprises a polypeptide having alpha-amylase        activity, wherein if said composition comprises a polypeptide        having alpha-amylase activity and the polypeptide is of fungal        origin, then the ratio of glucoamylase activity expressed as        AGU/g to alpha-amylase activity expressed as Fungal        alpha-Amylase Units FAU(A)/g in said composition is higher than        29, such as higher than 50, higher than 100, higher than 200,        higher than 300, higher than 400, higher than 500, higher than        600, higher than 800, higher than 1000, higher than 2000, higher        than 3000, higher than 4000, or such as higher than 5000, and        wherein if the polypeptide having alpha-amylase activity is of        bacterial origin, then the ratio of glucoamylase activity        expressed as AGU/g to alpha-amylase activity expressed as Kilo        Novo alpha-amylase Units (KNU)/g is higher than 29, such as        higher than 50, higher than 100, higher than 200, higher than        300, higher than 400, higher than 500, higher than 600, higher        than 800, higher than 1000, higher than 2000, higher than 3000,        higher than 4000, or such as higher than 5000.    -   23. The process according to any of items 14-21, wherein the        glucoamylase and the pullulanase added in step ii) are in a        composition as defined in any of items 1-13.    -   24. The process according to any of items 14-23, wherein said        incubation in step ii) of item 14 has a duration of from 2-35        hours, such as from 2-30 hours, from 2-28 hours, from 2-26        hours, from 2-24 hours, from 2-22 hours, from 2-20 hours, from        2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12 hours,        from 2-10 hours, from 2-8 hours, from 4-35 hours, from 4-30        hours, from 4-28 hours, from 4-26 hours, from 4-24, from 4-22        hours, from 4-20 hours, from 4-18 hours, from 4-16 hours, from        4-14 hours, from 4-12 hours, from 4-10 hours, from 4-8 hours,        from 10-35 hours, from 10-30 hours, from 10-28 hours, from 10-26        hours, from 10-24 hours, from 10-22 hours, from 10-20 hours,        from 10-18 hours, from 10-16 hours, from 10-14 hours, from 10-12        hours, from 14-35 hours, from 14-30 hours, from 14-28 hours,        from 14-26 hours, from 14-24, from 14-22 hours, from 14-20        hours, from 14-18 hours, from 14-16 hours, from 18-35 hours,        from 18-30 hours, from 18-28 hours, from 18-26 hours, from        18-24, from 18-22 hours, from 18-20 hours, from 20-35 hours,        from 20-30 hours, from 20-28 hours, from 20-26 hours, from        20-24, from 20-22 hours, form 22-35 hours, from 22-30 hours,        from 22-28 hours, from 22-26 hours, from 22-24 hours or such or        sus as a duration of 24 hours.    -   25. The process according to any of items 14-24, wherein said        incubation in step iii) of item 14 has a duration of from 2-94        hours, such as from 2-90 hours, from 2-80 hours, from 2-70        hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45        hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from        2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from        2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours,        from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-94        hours, from 4-90 hours, from 4-80 hours, from 4-70 hours, from        4-60, from 4-55 hours, from 4-50 hours, from 4-45 hours, from        4-40 hours, from 4-35 hours, from 4-30 hours, from 4-28 hours,        from 4-26 hours, from 4-24, from 4-22 hours, from 4-20 hours,        from 4-18 hours, from 4-16 hours, from 4-14 hours, from 4-12        hours, from 4-10 hours, from 4-8 hours, from 10-94 hours, from        10-90 hours, from 10-80 hours, from 10-70 hours, from 10-60,        from 10-55 hours, from 10-50 hours, from 10-45 hours, from 10-40        hours, from 10-35 hours, from 10-30 hours, from 10-28 hours,        from 10-26 hours, from 10-24, from 10-22 hours, from 10-20        hours, from 10-18 hours, from 10-16 hours, from 10-14 hours,        from 10-12 hours, from 14-94 hours, from 14-90 hours, from 14-80        hours, from 14-70 hours, from 14-60, from 14-55 hours, from        14-50 hours, from 14-45 hours, from 14-40 hours, from 14-35        hours, from 14-30 hours, from 14-28 hours, from 14-26 hours,        from 14-24, from 14-22 hours, from 14-20 hours, from 14-18        hours, from 14-16 hours, from 20-94 hours, from 20-90 hours,        from 20-80 hours, from 20-70 hours, from 20-60, from 20-55        hours, from 20-50 hours, from 20-45 hours, from 20-40 hours,        from 20-35 hours, from 20-30 hours, from 20-28 hours, from 20-26        hours, from 20-24, from 20-22 hours, from 24-94 hours, from        24-90 hours, from 24-80 hours, from 24-70 hours, from 24-60,        from 24-55 hours, from 24-50 hours, from 24-45 hours, from 24-40        hours, from 24-35 hours, from 24-30 hours, from 24-28 hours,        from 24-26 hours, from 30-94 hours, from 30-90 hours, from 30-80        hours, from 30-70 hours, from 30-60, from 30-55 hours, from        30-50 hours, from 30-45 hours, from 30-40 hours, from 30-35        hours, from 40-94 hours, from 40-90 hours, from 40-80 hours,        from 40-70 hours, from 40-60, from 40-55 hours, from 40-50        hours, from 40-45 hours, from 50-94 hours, from 50-90 hours,        from 50-80 hours, from 50-70 hours, from 50-60, from 60-94        hours, from 60-90 hours, from 60-80 hours, or such as a duration        of from 60-70 hours.    -   26. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 2-35        hours and said incubation in step iii) of item 14 has a duration        of from 2-94 hours, such as from 2-90 hours, from 2-80 hours,        from 2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours,        from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30        hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22        hours, from 2-20 hours, from 2-18 hours, from 2-16 hours, from        2-14 hours, from 2-12 hours, from 2-10 hours, from 2-8 hours,        from 4-94 hours, from 4-90 hours, from 4-80 hours, from 4-70        hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45        hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from        4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from        4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14 hours,        from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-94        hours, from 10-90 hours, from 10-80 hours, from 10-70 hours,        from 10-60, from 10-55 hours, from 10-50 hours, from 10-45        hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,        from 10-28 hours, from 10-26 hours, from 10-24, from 10-22        hours, from 10-20 hours, from 10-18 hours, from 10-16 hours,        from 10-14 hours, from 10-12 hours, from 14-94 hours, from 14-90        hours, from 14-80 hours, from 14-70 hours, from 14-60, from        14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40        hours, from 14-35 hours, from 14-30 hours, from 14-28 hours,        from 14-26 hours, from 14-24, from 14-22 hours, from 14-20        hours, from 14-18 hours, from 14-16 hours, from 20-94 hours,        from 20-90 hours, from 20-80 hours, from 20-70 hours, from        20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours,        from 20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28        hours, from 20-26 hours, from 20-24, from 20-22 hours, from        24-94 hours, from 24-90 hours, from 24-80 hours, from 24-70        hours, from 24-60, from 24-55 hours, from 24-50 hours, from        24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30        hours, from 24-28 hours, from 24-26 hours, from 30-94 hours,        from 30-90 hours, from 30-80 hours, from 30-70 hours, from        30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,        from 30-40 hours, from 30-35 hours, from 40-94 hours, from 40-90        hours, from 40-80 hours, from 40-70 hours, from 40-60, from        40-55 hours, from 40-50 hours, from 40-45 hours, from 50-94        hours, from 50-90 hours, from 50-80 hours, from 50-70 hours,        from 50-60, from 60-94 hours, from 60-90 hours, from 60-80        hours, or such as a duration of from 60-70 hours.    -   27. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 2-20        hours and said incubation in step iii) of item 14 has a duration        of from 2-94 hours, such as from 2-90 hours, from 2-80 hours,        from 2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours,        from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30        hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22        hours, from 2-20 hours, from 2-18 hours, from 2-16 hours, from        2-14 hours, from 2-12 hours, from 2-10 hours, from 2-8 hours,        from 4-94 hours, from 4-90 hours, from 4-80 hours, from 4-70        hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45        hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from        4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from        4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14 hours,        from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-94        hours, from 10-90 hours, from 10-80 hours, from 10-70 hours,        from 10-60, from 10-55 hours, from 10-50 hours, from 10-45        hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,        from 10-28 hours, from 10-26 hours, from 10-24, from 10-22        hours, from 10-20 hours, from 10-18 hours, from 10-16 hours,        from 10-14 hours, from 10-12 hours, from 14-94 hours, from 14-90        hours, from 14-80 hours, from 14-70 hours, from 14-60, from        14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40        hours, from 14-35 hours, from 14-30 hours, from 14-28 hours,        from 14-26 hours, from 14-24, from 14-22 hours, from 14-20        hours, from 14-18 hours, from 14-16 hours, from 20-94 hours,        from 20-90 hours, from 20-80 hours, from 20-70 hours, from        20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours,        from 20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28        hours, from 20-26 hours, from 20-24, from 20-22 hours, from        24-94 hours, from 24-90 hours, from 24-80 hours, from 24-70        hours, from 24-60, from 24-55 hours, from 24-50 hours, from        24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30        hours, from 24-28 hours, from 24-26 hours, from 30-94 hours,        from 30-90 hours, from 30-80 hours, from 30-70 hours, from        30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,        from 30-40 hours, from 30-35 hours, from 40-94 hours, from 40-90        hours, from 40-80 hours, from 40-70 hours, from 40-60, from        40-55 hours, from 40-50 hours, from 40-45 hours, from 50-94        hours, from 50-90 hours, from 50-80 hours, from 50-70 hours,        from 50-60, from 60-94 hours, from 60-90 hours, from 60-80        hours, or such as a duration of from 60-70 hours.    -   28. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 4-35        hours and said incubation in step iii) of item 14 has a duration        of from 2-92 hours, such as from 2-90 hours, from 2-80 hours,        from 2-70 hours, from 2-60, from 2-55 hours, from 2-50 hours,        from 2-45 hours, from 2-40 hours, from 2-35 hours, from 2-30        hours, from 2-28 hours, from 2-26 hours, from 2-24, from 2-22        hours, from 2-20 hours, from 2-18 hours, from 2-16 hours, from        2-14 hours, from 2-12 hours, from 2-10 hours, from 2-8 hours,        from 4-92 hours, from 4-90 hours, from 4-80 hours, from 4-70        hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45        hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from        4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from        4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14 hours,        from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-92        hours, from 10-90 hours, from 10-80 hours, from 10-70 hours,        from 10-60, from 10-55 hours, from 10-50 hours, from 10-45        hours, from 10-40 hours, from 10-35 hours, from 10-30 hours,        from 10-28 hours, from 10-26 hours, from 10-24, from 10-22        hours, from 10-20 hours, from 10-18 hours, from 10-16 hours,        from 10-14 hours, from 10-12 hours, from 14-92 hours, from 14-90        hours, from 14-80 hours, from 14-70 hours, from 14-60, from        14-55 hours, from 14-50 hours, from 14-45 hours, from 14-40        hours, from 14-35 hours, from 14-30 hours, from 14-28 hours,        from 14-26 hours, from 14-24, from 14-22 hours, from 14-20        hours, from 14-18 hours, from 14-16 hours, from 20-92 hours,        from 20-90 hours, from 20-80 hours, from 20-70 hours, from        20-60, from 20-55 hours, from 20-50 hours, from 20-45 hours,        from 20-40 hours, from 20-35 hours, from 20-30 hours, from 20-28        hours, from 20-26 hours, from 20-24, from 20-22 hours, from        24-92 hours, from 24-90 hours, from 24-80 hours, from 24-70        hours, from 24-60, from 24-55 hours, from 24-50 hours, from        24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30        hours, from 24-28 hours, from 24-26 hours, from 30-92 hours,        from 30-90 hours, from 30-80 hours, from 30-70 hours, from        30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,        from 30-40 hours, from 30-35 hours, from 40-92 hours, from 40-90        hours, from 40-80 hours, from 40-70 hours, from 40-60, from        40-55 hours, from 40-50 hours, from 40-45 hours, from 50-92        hours, from 50-90 hours, from 50-80 hours, from 50-70 hours,        from 50-60, from 60-94 hours, from 60-90 hours, from 60-80        hours, or such as a duration of from 60-70 hours.    -   29. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 4-22        hours, and said incubation in step iii) of item 14 has a        duration of from 2-92 hours, such as from 2-90 hours, from 2-80        hours, from 2-70 hours, from 2-60, from 2-55 hours, from 2-50        hours, from 2-45 hours, from 2-40 hours, from 2-35 hours, from        2-30 hours, from 2-28 hours, from 2-26 hours, from 2-24, from        2-22 hours, from 2-20 hours, from 2-18 hours, from 2-16 hours,        from 2-14 hours, from 2-12 hours, from 2-10 hours, from 2-8        hours, from 4-92 hours, from 4-90 hours, from 4-80 hours, from        4-70 hours, from 4-60, from 4-55 hours, from 4-50 hours, from        4-45 hours, from 4-40 hours, from 4-35 hours, from 4-30 hours,        from 4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours,        from 4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14        hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from        10-92 hours, from 10-90 hours, from 10-80 hours, from 10-70        hours, from 10-60, from 10-55 hours, from 10-50 hours, from        10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30        hours, from 10-28 hours, from 10-26 hours, from 10-24, from        10-22 hours, from 10-20 hours, from 10-18 hours, from 10-16        hours, from 10-14 hours, from 10-12 hours, from 14-92 hours,        from 14-90 hours, from 14-80 hours, from 14-70 hours, from        14-60, from 14-55 hours, from 14-50 hours, from 14-45 hours,        from 14-40 hours, from 14-35 hours, from 14-30 hours, from 14-28        hours, from 14-26 hours, from 14-24, from 14-22 hours, from        14-20 hours, from 14-18 hours, from 14-16 hours, from 20-92        hours, from 20-90 hours, from 20-80 hours, from 20-70 hours,        from 20-60, from 20-55 hours, from 20-50 hours, from 20-45        hours, from 20-40 hours, from 20-35 hours, from 20-30 hours,        from 20-28 hours, from 20-26 hours, from 20-24, from 20-22        hours, from 24-92 hours, from 24-90 hours, from 24-80 hours,        from 24-70 hours, from 24-60, from 24-55 hours, from 24-50        hours, from 24-45 hours, from 24-40 hours, from 24-35 hours,        from 24-30 hours, from 24-28 hours, from 24-26 hours, from 30-92        hours, from 30-90 hours, from 30-80 hours, from 30-70 hours,        from 30-60, from 30-55 hours, from 30-50 hours, from 30-45        hours, from 30-40 hours, from 30-35 hours, from 40-92 hours,        from 40-90 hours, from 40-80 hours, from 40-70 hours, from        40-60, from 40-55 hours, from 40-50 hours, from 40-45 hours,        from 50-92 hours, from 50-90 hours, from 50-80 hours, from 50-70        hours, from 50-60, from 60-94 hours, from 60-90 hours, from        60-80 hours, or such as a duration of from 60-70 hours.    -   30. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 10-35        hours, and said incubation in step iii) of item 14 has a        duration of from 2-86 hours, such as from 2-80 hours, from 2-70        hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45        hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from        2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from        2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours,        from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-86        hours, from 4-80 hours, from 4-70 hours, from 4-60, from 4-55        hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from        4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26 hours,        from 4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours,        from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10        hours, from 4-8 hours, from 10-86 hours, from 10-80 hours, from        10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,        from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30        hours, from 10-28 hours, from 10-26 hours, from 10-24, from        10-22 hours, from 10-20 hours, from 10-18 hours, from 10-16        hours, from 10-14 hours, from 10-12 hours, from 14-86 hours,        from 14-80 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-86 hours, from 20-80        hours, from 20-70 hours, from 20-60, from 20-55 hours, from        20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35        hours, from 20-30 hours, from 20-28 hours, from 20-26 hours,        from 20-24, from 20-22 hours, from 25-86 hours, from 20-80        hours, from 20-70 hours, from 20-60, from 20-55 hours, from        20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35        hours, from 20-30 hours, from 20-28 hours, from 20-26 hours,        from 20-24, from 20-22 hours, from 24-86 hours, from 24-80        hours, from 24-70 hours, from 24-60, from 24-55 hours, from        24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35        hours, from 24-30 hours, from 24-28 hours, from 24-26 hours,        from 30-86 hours, from 30-80 hours, from 30-70 hours, from        30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,        from 30-40 hours, from 30-35 hours, from 40-86 hours, from 40-80        hours, from 40-70 hours, from 40-60, from 40-55 hours, from        40-50 hours, from 40-45 hours, from 50-86 hours, from 50-80        hours, from 50-70 hours, from 50-60, from 60-94 hours, from        60-90 hours, from 60-80 hours, or such as a duration of from        60-70 hours.    -   31. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 10-24        hours, and said incubation in step iii) of item 14 has a        duration of from 2-86 hours, such as from 2-80 hours, from 2-70        hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45        hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from        2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from        2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours,        from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-86        hours, from 4-80 hours, from 4-70 hours, from 4-60, from 4-55        hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from        4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26 hours,        from 4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours,        from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10        hours, from 4-8 hours, from 10-86 hours, from 10-80 hours, from        10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,        from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30        hours, from 10-28 hours, from 10-26 hours, from 10-24, from        10-22 hours, from 10-20 hours, from 10-18 hours, from 10-16        hours, from 10-14 hours, from 10-12 hours, from 14-86 hours,        from 14-80 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-86 hours, from 20-80        hours, from 20-70 hours, from 20-60, from 20-55 hours, from        20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35        hours, from 20-30 hours, from 20-28 hours, from 20-26 hours,        from 20-24, from 20-22 hours, from 25-86 hours, from 20-80        hours, from 20-70 hours, from 20-60, from 20-55 hours, from        20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35        hours, from 20-30 hours, from 20-28 hours, from 20-26 hours,        from 20-24, from 20-22 hours, from 24-86 hours, from 24-80        hours, from 24-70 hours, from 24-60, from 24-55 hours, from        24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35        hours, from 24-30 hours, from 24-28 hours, from 24-26 hours,        from 30-86 hours, from 30-80 hours, from 30-70 hours, from        30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,        from 30-40 hours, from 30-35 hours, from 40-86 hours, from 40-80        hours, from 40-70 hours, from 40-60, from 40-55 hours, from        40-50 hours, from 40-45 hours, from 50-86 hours, from 50-80        hours, from 50-70 hours, from 50-60, from 60-94 hours, from        60-90 hours, from 60-80 hours, or such as a duration of from        60-70 hours.    -   32. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 14-35        hours, and said incubation in step iii) of item 14 has a        duration of from 2-70 hours, such as from 2-60, from 2-55 hours,        from 2-50 hours, from 2-45 hours, from 2-40 hours, from 2-35        hours, from 2-30 hours, from 2-28 hours, from 2-26 hours, from        2-24, from 2-22 hours, from 2-20 hours, from 2-18 hours, from        2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10 hours,        from 2-8 hours, from 4-70 hours, from 4-60, from 4-55 hours,        from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35        hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from        4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours, from        4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10 hours,        from 4-8 hours, from 10-70 hours, from 10-60, from 10-55 hours,        from 10-50 hours, from 10-45 hours, from 10-40 hours, from 10-35        hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,        from 10-24, from 10-22 hours, from 10-20 hours, from 10-18        hours, from 10-16 hours, from 10-14 hours, from 10-12 hours,        from 14-70 hours, from 14-60, from 14-55 hours, from 14-50        hours, from 14-45 hours, from 14-40 hours, from 14-35 hours,        from 14-30 hours, from 14-28 hours, from 14-26 hours, from        14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,        from 14-16 hours, from 20-70 hours, from 20-60, from 20-55        hours, from 20-50 hours, from 20-45 hours, from 20-40 hours,        from 20-35 hours, from 20-30 hours, from 20-28 hours, from 20-26        hours, from 20-24, from 20-22 hours, from 25-94 hours, from        25-90 hours, from 20-80 hours, from 20-70 hours, from 20-60,        from 20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40        hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,        from 20-26 hours, from 20-24, from 20-22 hours, from 24-70        hours, from 24-60, from 24-55 hours, from 24-50 hours, from        24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30        hours, from 24-28 hours, from 24-26 hours, from 30-70 hours,        from 30-60, from 30-55 hours, from 30-50 hours, from 30-45        hours, from 30-40 hours, from 30-35 hours, from 40-70 hours,        from 40-60, from 40-55 hours, from 40-50 hours, from 40-45        hours, from 50-70 hours, from 50-60, from 60-94 hours, from        60-90 hours, from 60-80 hours, or such as a duration of from        60-70 hours.    -   33. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 14-26        hours, and said incubation in step iii) of item 14 has a        duration of from 2-70 hours, such as from 2-60, from 2-55 hours,        from 2-50 hours, from 2-45 hours, from 2-40 hours, from 2-35        hours, from 2-30 hours, from 2-28 hours, from 2-26 hours, from        2-24, from 2-22 hours, from 2-20 hours, from 2-18 hours, from        2-16 hours, from 2-14 hours, from 2-12 hours, from 2-10 hours,        from 2-8 hours, from 4-70 hours, from 4-60, from 4-55 hours,        from 4-50 hours, from 4-45 hours, from 4-40 hours, from 4-35        hours, from 4-30 hours, from 4-28 hours, from 4-26 hours, from        4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours, from        4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10 hours,        from 4-8 hours, from 10-70 hours, from 10-60, from 10-55 hours,        from 10-50 hours, from 10-45 hours, from 10-40 hours, from 10-35        hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,        from 10-24, from 10-22 hours, from 10-20 hours, from 10-18        hours, from 10-16 hours, from 10-14 hours, from 10-12 hours,        from 14-70 hours, from 14-60, from 14-55 hours, from 14-50        hours, from 14-45 hours, from 14-40 hours, from 14-35 hours,        from 14-30 hours, from 14-28 hours, from 14-26 hours, from        14-24, from 14-22 hours, from 14-20 hours, from 14-18 hours,        from 14-16 hours, from 20-70 hours, from 20-60, from 20-55        hours, from 20-50 hours, from 20-45 hours, from 20-40 hours,        from 20-35 hours, from 20-30 hours, from 20-28 hours, from 20-26        hours, from 20-24, from 20-22 hours, from 25-94 hours, from        25-90 hours, from 20-80 hours, from 20-70 hours, from 20-60,        from 20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40        hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,        from 20-26 hours, from 20-24 hours, from 20-22 hours, from 24-70        hours, from 24-60, from 24-55 hours, from 24-50 hours, from        24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30        hours, from 24-28 hours, from 24-26 hours, from 30-70 hours,        from 30-60, from 30-55 hours, from 30-50 hours, from 30-45        hours, from 30-40 hours, from 30-35 hours, from 40-70 hours,        from 40-60, from 40-55 hours, from 40-50 hours, from 40-45        hours, from 50-70 hours, from 50-60, from 60-94 hours, from        60-90 hours, from 60-80 hours, or such as a duration of from        60-70 hours.    -   34. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 18-35        hours, and said incubation in step iii) of item 14 has a        duration of from 2-78 hours, such as from 2-74 hours, from 2-70        hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45        hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from        2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from        2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours,        from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-78        hours, from 4-74 hours, from 4-70 hours, from 4-60, from 4-55        hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from        4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26 hours,        from 4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours,        from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10        hours, from 4-8 hours, from 10-76 hours, from 10-74 hours, from        10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,        from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30        hours, from 10-28 hours, from 10-26 hours, from 10-24, from        10-22 hours, from 10-20 hours, from 10-18 hours, from 10-16        hours, from 10-14 hours, from 10-12 hours, from 14-78 hours,        from 14-74 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-78 hours, from 20-74        hours, from 20-80 hours, from 20-70 hours, from 20-60, from        20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40        hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,        from 20-26 hours, from 20-24, from 20-22 hours, from 24-78        hours, from 24-76 hours, from 24-70 hours, from 24-60, from        24-55 hours, from 24-50 hours, from 24-45 hours, from 24-40        hours, from 24-35 hours, from 24-30 hours, from 24-28 hours,        from 24-26 hours, from 30-78 hours, from 30-74 hours, from 30-70        hours, from 30-60, from 30-55 hours, from 30-50 hours, from        30-45 hours, from 30-40 hours, from 30-35 hours, from 40-76        hours, from 40-74 hours, from 40-70 hours, from 40-60, from        40-55 hours, from 40-50 hours, from 40-45 hours, from 50-78        hours, from 50-74 hours, from 50-70 hours, from 50-60, from        60-94 hours, from 60-90 hours, from 60-80 hours, or such as a        duration of from 60-70 hours.    -   35. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 18-28        hours, and said incubation in step iii) of item 14 has a        duration of from 2-78 hours, such as from 2-74 hours, from 2-70        hours, from 2-60, from 2-55 hours, from 2-50 hours, from 2-45        hours, from 2-40 hours, from 2-35 hours, from 2-30 hours, from        2-28 hours, from 2-26 hours, from 2-24, from 2-22 hours, from        2-20 hours, from 2-18 hours, from 2-16 hours, from 2-14 hours,        from 2-12 hours, from 2-10 hours, from 2-8 hours, from 4-78        hours, from 4-74 hours, from 4-70 hours, from 4-60, from 4-55        hours, from 4-50 hours, from 4-45 hours, from 4-40 hours, from        4-35 hours, from 4-30 hours, from 4-28 hours, from 4-26 hours,        from 4-24, from 4-22 hours, from 4-20 hours, from 4-18 hours,        from 4-16 hours, from 4-14 hours, from 4-12 hours, from 4-10        hours, from 4-8 hours, from 10-76 hours, from 10-74 hours, from        10-70 hours, from 10-60, from 10-55 hours, from 10-50 hours,        from 10-45 hours, from 10-40 hours, from 10-35 hours, from 10-30        hours, from 10-28 hours, from 10-26 hours, from 10-24, from        10-22 hours, from 10-20 hours, from 10-18 hours, from 10-16        hours, from 10-14 hours, from 10-12 hours, from 14-78 hours,        from 14-74 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-78 hours, from 20-74        hours, from 20-80 hours, from 20-70 hours, from 20-60, from        20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40        hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,        from 20-26 hours, from 20-24, from 20-22 hours, from 24-78        hours, from 24-76 hours, from 24-70 hours, from 24-60, from        24-55 hours, from 24-50 hours, from 24-45 hours, from 24-40        hours, from 24-35 hours, from 24-30 hours, from 24-28 hours,        from 24-26 hours, from 30-78 hours, from 30-74 hours, from 30-70        hours, from 30-60, from 30-55 hours, from 30-50 hours, from        30-45 hours, from 30-40 hours, from 30-35 hours, from 40-76        hours, from 40-74 hours, from 40-70 hours, from 40-60, from        40-55 hours, from 40-50 hours, from 40-45 hours, from 50-78        hours, from 50-74 hours, from 50-70 hours, from 50-60, from        60-94 hours, from 60-90 hours, from 60-80 hours, or such as a        duration of from 60-70 hours.    -   36. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 20-35        hours, and said incubation in step iii) of item 14 has a        duration of from 2-76 hours, such as from 2-70 hours, from 2-60,        from 2-55 hours, from 2-50 hours, from 2-45 hours, from 2-40        hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from        2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours, from        2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12 hours,        from 2-10 hours, from 2-8 hours, from 4-76 hours, from 4-70        hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45        hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from        4-28 hours, from 4-26 hours, from 4-24 hours, from 4-22 hours,        from 4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14        hours, from 4-12 hours, from 4-10 hours, from 4-8 hours, from        10-76 hours, from 10-70 hours, from 10-60, from 10-55 hours,        from 10-50 hours, from 10-45 hours, from 10-40 hours, from 10-35        hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,        from 10-24 ours, from 10-22 hours, from 10-20 hours, from 10-18        hours, from 10-16 hours, from 10-14 hours, from 10-12 hours,        from 14-76 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-76 hours, from 20-70        hours, from 20-60, from 20-55 hours, from 20-50 hours, from        20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30        hours, from 20-28 hours, from 20-26 hours, from 20-24, from        20-22 hours, from 25-76 hours, from 20-70 hours, from 20-60,        from 20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40        hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,        from 20-26 hours, from 20-24, from 20-22 hours, from 24-76        hours, from 24-70 hours, from 24-60, from 24-55 hours, from        24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35        hours, from 24-30 hours, from 24-28 hours, from 24-26 hours,        from 30-76 hours, from 30-70 hours, from 30-60, from 30-55        hours, from 30-50 hours, from 30-45 hours, from 30-40 hours,        from 30-35 hours, from 40-76 hours, from 40-70 hours, from        40-60, from 40-55 hours, from 40-50 hours, from 40-45 hours,        from 50-76 hours, from 50-70 hours, from 50-60, from 60-94        hours, from 60-90 hours, from 60-80 hours, or such as a duration        of from 60-70 hours.    -   37. The process according to any of items 14-22, wherein said        incubation in step ii) of item 14 has a duration of from 20-30        hours, and said incubation in step iii) of item 14 has a        duration of from 2-76 hours, such as from 2-70 hours, from 2-60,        from 2-55 hours, from 2-50 hours, from 2-45 hours, from 2-40        hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from        2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours, from        2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12 hours,        from 2-10 hours, from 2-8 hours, from 4-76 hours, from 4-70        hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45        hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from        4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from        4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14 hours,        from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-76        hours, from 10-70 hours, from 10-60, from 10-55 hours, from        10-50 hours, from 10-45 hours, from 10-40 hours, from 10-35        hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,        from 10-24 hours, from 10-22 hours, from 10-20 hours, from 10-18        hours, from 10-16 hours, from 10-14 hours, from 10-12 hours,        from 14-76 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-76 hours, from 20-70        hours, from 20-60, from 20-55 hours, from 20-50 hours, from        20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30        hours, from 20-28 hours, from 20-26 hours, from 20-24, from        20-22 hours, from 25-76 hours, from 20-70 hours, from 20-60,        from 20-55 hours, from 20-50 hours, from 20-45 hours, from 20-40        hours, from 20-35 hours, from 20-30 hours, from 20-28 hours,        from 20-26 hours, from 20-24, from 20-22 hours, from 24-76        hours, from 24-70 hours, from 24-60, from 24-55 hours, from        24-50 hours, from 24-45 hours, from 24-40 hours, from 24-35        hours, from 24-30 hours, from 24-28 hours, from 24-26 hours,        from 30-76 hours, from 30-70 hours, from 30-60, from 30-55        hours, from 30-50 hours, from 30-45 hours, from 30-40 hours,        from 30-35 hours, from 40-76 hours, from 40-70 hours, from        40-60, from 40-55 hours, from 40-50 hours, from 40-45 hours,        from 50-76 hours, from 50-70 hours, from 50-60, from 60-94        hours, from 60-90 hours, from 60-80 hours, or such as a duration        of from 60-70 hours.    -   38. The process according to any of items 14-25, wherein said        incubation in step ii) of item 14 has a duration of from 22-35        hours, and said incubation in step iii) of item 14 has a        duration of from 2-74 hours, such as from 2-70 hours, from 2-60,        from 2-55 hours, from 2-50 hours, from 2-45 hours, from 2-40        hours, from 2-35 hours, from 2-30 hours, from 2-28 hours, from        2-26 hours, from 2-24, from 2-22 hours, from 2-20 hours, from        2-18 hours, from 2-16 hours, from 2-14 hours, from 2-12 hours,        from 2-10 hours, from 2-8 hours, from 4-74 hours, from 4-70        hours, from 4-60, from 4-55 hours, from 4-50 hours, from 4-45        hours, from 4-40 hours, from 4-35 hours, from 4-30 hours, from        4-28 hours, from 4-26 hours, from 4-24, from 4-22 hours, from        4-20 hours, from 4-18 hours, from 4-16 hours, from 4-14 hours,        from 4-12 hours, from 4-10 hours, from 4-8 hours, from 10-74        hours, from 10-70 hours, from 10-60, from 10-55 hours, from        10-50 hours, from 10-45 hours, from 10-40 hours, from 10-35        hours, from 10-30 hours, from 10-28 hours, from 10-26 hours,        from 10-24 hours, from 10-22 hours, from 10-20 hours, from 10-18        hours, from 10-16 hours, from 10-14 hours, from 10-12 hours,        from 14-74 hours, from 14-70 hours, from 14-60, from 14-55        hours, from 14-50 hours, from 14-45 hours, from 14-40 hours,        from 14-35 hours, from 14-30 hours, from 14-28 hours, from 14-26        hours, from 14-24, from 14-22 hours, from 14-20 hours, from        14-18 hours, from 14-16 hours, from 20-74 hours, from 20-70        hours, from 20-60, from 20-55 hours, from 20-50 hours, from        20-45 hours, from 20-40 hours, from 20-35 hours, from 20-30        hours, from 20-28 hours, from 20-26 hours, from 20-24, from        20-22 hours, from 25-94 hours, from 25-90 hours, from 20-80        hours, from 20-70 hours, from 20-60, from 20-55 hours, from        20-50 hours, from 20-45 hours, from 20-40 hours, from 20-35        hours, from 20-30 hours, from 20-28 hours, from 20-26 hours,        from 20-24, from 20-22 hours, from 24-74 hours, from 24-70        hours, from 24-60, from 24-55 hours, from 24-50 hours, from        24-45 hours, from 24-40 hours, from 24-35 hours, from 24-30        hours, from 24-28 hours, from 24-26 hours, from 30-94 hours,        from 30-90 hours, from 30-80 hours, from 30-70 hours, from        30-60, from 30-55 hours, from 30-50 hours, from 30-45 hours,        from 30-40 hours, from 30-35 hours, from 40-74 hours, from 40-70        hours, from 40-60, from 40-55 hours, from 40-50 hours, from        40-45 hours, from 50-94 hours, from 50-90 hours, from 50-80        hours, from 50-70 hours, from 50-60, from 60-74 hours, or such        as a duration of from 60-70 hours.    -   39. The process according to any of items 14-38, wherein the        incubation in step iii) of item 14 is allowed to proceed until        the amount of glucose (% DX) in said composition has reached a        level corresponding to 95% (w/w) or more, such as 95.5% (w/w) or        more, 95.75% (w/w) or more, 96% (w/w) or more, 96.25% (w/w) or        more, or such as 96.75% (w/w) or more.    -   40. The process according to any of items 14-39, wherein the        duration of the process from addition of said a glucoamylase        (AMG) and said pullulanase to the composition comprising        liquefied starch until termination of the starch        hydrolysis/saccharification is from 24-96 hours, such as from        24-72 hours, from 24-64 hours, from 24-48 hours, from 24-36        hours, from 24-32 hours, from 32-96 hours, from 32-72 hours,        from 32-64 hours, from 32-48 hours, from 32-36 hours, from 36-96        hours, from 36-72 hours, from 36-64 hours, from 36-48 hours,        from 48-96 hours, such as from 48-72 hours, from 48-64 hours,        from 56-96 hours, such as from 56-72 hours, or such as from        56-64 hours,    -   41. The process according to any of items 14-40, wherein the        starch hydrolysis/saccharification takes place at a pH which is        within the range of 3.5-5.0, such as at pH in the range of        4.0-4.5, and at a temperature, which is within the range of        59−70° C., such as in the range of 59−65° C. or such as in the        range of 59−62° C.    -   42. The process according to any of items 14-41, wherein the        composition comprising liquefied starch provided in step i) of        item 14 contains from 25-45% dry solids (% DS), such as from        25-40% DS, from 28-38% DS, from 30-38% DS, or such as from        33-36% DS.    -   43. The process according to any of items 14-42, wherein the        alpha amylase is selected from Bacillus licheniformis, Bacillus        stearothermophilus, Aspergillus niger, Aspergillus terreus or        Rhizomucor pusillus alpha amylases, the glucoamylase is selected        from Aspergillus niger, Aspergillus fumigatus, Talaromyces        emersonii, Trametes cingulata, Trichoderma reesei, Humicloa        insolens and Gloeophyllum trabeum glucoamylases, and the        pullulanase is selected from Bacillus deramificans or Bacillus        acidopullulyticus pullulanases.    -   44. The process according to any of items 14-43, wherein the        glucoamylase comprises/consists essentially of/consists of an        amino acid sequence selected from the group consisting of        -   iv) The amino acid sequence set forth in any of SEQ ID NO:            2-12 and 27;        -   v) A subsequence of the amino acid sequence set forth in any            one of SEQ ID NOs: 2-12 and 27;        -   vi) An amino acid sequence, which has at least 70%, such as            at least 75%, at least 80%, at least 85%, at least 90%, at            least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 96%, at least 97%, at least 98%, at            least 99%, such as at least 99.5% sequence identity to any            one of the amino acids sequences set forth in i) and ii).    -   45. The process according to any of items 14-44, wherein the        alpha-amylase comprises/consists essentially of/consists of an        amino acid sequence selected from the group consisting of        -   iv) The amino acid sequence set forth in any of SEQ ID NOs:            1 and 17-26;        -   v) A subsequence of the amino acid sequence set forth in any            one of SEQ ID NOs: 1 and 17-26;        -   vi) An amino acid sequence, which has at least 70%, such as            at least 75%, at least 80%, at least 85%, at least 90%, at            least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 96%, at least 97%, at least 98%, at            least 99%, such as at least 99.5% sequence identity to any            one of the amino acids sequences set forth in i) and ii).    -   46. The process according to any of items 14-45, wherein the        pullulanase comprises/consists essentially of/consists of an        amino acid sequence selected from the group consisting of        -   iv) The amino acid sequence set forth in any one of SEQ ID            NOs: 13-16;        -   v) A subsequence of the amino acid sequence set forth in any            one of SEQ ID NOs: 13-16;        -   vi) An amino acid sequence, which has at least 70%, such as            at least 75%, at least 80%, at least 85%, at least 90%, at            least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 96%, at least 97%, at least 98%, at            least 99%, such as at least 99.5% sequence identity to any            one of the amino acids sequences set forth in i) and ii).    -   47. A process for producing glucose syrup from liquefied starch,        comprising contacting the liquefied starch with a composition        according to any of items 1-13.    -   48. The process according to item 47, wherein said liquefied        starch contains from 25-45% dry solids (% DS), such as from        25-40% DS, from 30-38% DS, from 32-38% DS, or such as from        34-36% DS.    -   49. The process according to any of items 47-48, said process        comprising subjecting the liquified starch to incubation under        conditions allowing starch hydrolysis/saccharification.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1

Maltodextrin powder from corn starch liquefaction was dissolved in waterwhile heating to make slurry at 34.2% dry solids. The solid content ofthe slurry was measured using Refractive index measurement showing1.39221. The slurry was adjusted to a pH of 4.3 using a 1M Hydrochloricacid solution. 18 gram aliquots of this slurry were added to 18 glassreaction scintillation vials with septum cap closures and were insertedin a heating block to be heated to a temperature of 61° C. Each vial wasgiven an enzyme dosage based on the table below and additional water wasadded to each vial to reach a target dry solid of 33%. 1.5 mL sampleswere taken via needles through the septum from each vial at differenttime points (36 hour and 48 hours) and were deactivated at 105° C. for 5minutes. 1 mL of each deactivated sample was diluted with 4 mL deionizedwater. The diluted samples were evaluated using a HPLC method DP1-4 formeasuring dextrose purity (% DP1 or % DX, % DP2, % DP3 and % DP4+).

Results from Table 1 show the higher the NPUN/AGU ratio, the higher thepercent dextrose.

Table 1 shows that constant AGU dose of 0.25 AGU/gDS, when the ratio ofNPUN to AGU (NPUN/AGU) increased in the blend from 10.1 to 30.2, anincrease in % DX at peak was observed (96.7 to 97.0% DX). The newlycreated blends with NPUN/AGU of 9 or more show 0.7 to 1% DX improvementover commercial products. When the NPUN/g DS dose in the blend was keptas constant, by varying the AGU/gDS dose, higher % DX was not observed,only the speed of reaction was affected. The benefit in % DX in blendswith NPUN/AGU>9 is not only due to use of a lower isomaltose forming AMG(JGA98) showing lower DP2 of 2.1-2.2% at 36 hours but also is due tolower % DP3 and DP4+ at 36 hours.

TABLE 1 Dextrose content NPUN/ AGU/ NPUN/ AGU % DX % DX % DP2 % DP3 %DP4+ Enzyme gDS gDS ratio at 36 hr at 48 hr at 36 hr at 36 hr at 36 hrDextrozyme DX 2X 0.18 0.36 2 96.0 96.0 2.4 0.8 0.8 Dextrozyme DX Plus0.18 0.72 4 96.1 95.9 2.4 0.8 0.7 AMG (SEQ ID NO: 1) + 0.28 2.52 9 96.696.7 2.2 0.5 0.7 Pullulanase (SEQ ID NO: 13 (0.28/2.52) AMG (SEQ IDNO: 1) + 0.25 2.52 10.1 96.5 96.7 2.2 0.5 0.8 Pullulanase (SEQ ID NO: 13(0.25/2.52) AMG (SEQ ID NO: 1) + 0.23 2.52 11.0 96.5 96.7 2.1 0.5 0.9Pullulanase (SEQ ID NO: 13 (0.23/2.52) AMG (SEQ ID NO: 1) + 0.25 3.614.4 96.5 96.7 2.2 0.5 0.7 Pullulanase (SEQ ID NO: 13 (0.25/3.6) AMG(SEQ ID NO: 1) + 0.25 5.04 20.2 96.9 96.9 2.2 0.5 0.5 Pullulanase (SEQID NO: 13 (0.25/5.04) AMG (SEQ ID NO: 1) + 0.25 7.56 30.2 97.0 96.7 2.10.5 0.4 Pullulanase (SEQ ID NO: 13 (0.25/7.56)

Example 2

Maltodextrin powder from corn starch liquefaction was dissolved in waterwhile heating to make slurry starch slurry at 37.8% dry solids. Thesolid content of the slurry was measured using Refractive indexmeasurement showing 1.39964. The slurry was adjusted to a pH of 4.3using a 1M Hydrochloric acid solution. 18 gram aliquots of this slurrywere added to 18 glass reaction scintillation vials with septum capclosures and were inserted in a heating block to be heated to atemperature of 61° C. Each vial was given an enzyme dosage based on thetable below and additional water was added to each vial to reach atarget dry solid of 30, 33 or 36%. 1.5 mL samples were taken via needlesthrough the septum from each vial at different time points and weredeactivated at 105° C. for 5 minutes. 1 mL of each deactivated samplewas diluted with 4 mL deionized water. The diluted samples wereevaluated using a HPLC method DP1-4 for measuring dextrose purity (% DP1or % DX).

Table 2 shows the dextrose purity at 48 hours at the three differentsolids. The new enzyme blend shows 0.8-0.9% DX improvement overcommercial enzyme blend at all solids tested. This results show thatwith new enzyme blend at the same solids higher DX (0.8-0.9% DXimprovement) can be achieved. Also similar dextrose purity can beachieved at higher solids. For example using the new enzyme blend a % DXof 96.5% DX can be achieved at 36% DS instead of 30% DS using DextrozymeDX 2×.

TABLE 2 Dextrose purity Dose % DX at 48 hours (Peak time) Enzyme AGU/gDSNPUN/gDS NPUN/AGU 30% DS 33% DS 36% DS Dextrozyme DX 0.18 0.36 2 96.596.1 95.6 2X AMG (SEQ ID 0.25 2.52 10.08 97.4 96.9 96.5 NO: 12) +Pullulanase (SEQ ID NO: 13)

Example 3

Saccharification by enzyme blends with delayed dosing of a fungalalpha-amylase, (SEQ ID NO: 15), was evaluated to see how delayed dosingof alpha-amylase affects the DX and the speed of saccharification.

Materials and Methods

Saccharification test was carried out using a Gilson Liquid handler,with which 36% DS of DE11 maltodextrin made via Liquozyme Supra from waspH adjusted to 4.3 and was saccharified at 60° C. Prior tosaccharification any residual alpha-amylase activity in the maltodextrinwas inactivated by heat treatment. At different time points, the ratioof DX (glucose), DP2, DP3 and DP4+ of the syrup was analyzed by HPLC.

The initial enzyme dosages are shown in Table 1. “Benchmark” is aconventional enzyme blend for saccharification, containing glucoamylase,pullulanase and apha-amylase, the latter being a side activity of theglucoamylase. “AMG+Pul” is a blend of glucoamylase with no alpha-amylaseside activity and pullulanase. The sample volume increased by thealpha-amylase spike was only 0.2% so that the change in DS isnegligible.

After 24 h incubation with AMG+Pul blend, purified alpha-amylase (SEQ IDNO: 15) was dosed to be 0.01 FAU(A)/gDS, and compared with the benchmarkenzymes. The dosages and the timings of enzymes are shown in Table 1.

TABLE 3 Enzyme dosages Alpha- Alpha- Pullulanase amylase amylase SEQ IDSEQ ID NO: 1 SEQ ID NO: NO: 13 (side activity) 15 spiked Spiked EnzymeGlucoamylase [AGU/ [NPUN [FAU(A)/ [FAU(A)/ Spiked Sample blend (AMG)gDS] (XP)/gDS] gDS] gDS] at 1 Bench- SEQ ID NO: 1 0.18 0.36 0.045 — —mark 2 AMG + Pul SEQ ID NO: 12 0.194 2.52 — — — blend 3 AMG + Pul SEQ IDNO: 12 0.194 2.52 — 0.01 24 h blend, AA spiked

Results

The DX curves from two experiments (FIG. 1) and the summary table (Table2) show that the spike of alpha-amylase at 24 h both improved thesaccharification speed and the peak DX significantly, compared toAMG+Pul blend. When compared at 48 h (saccharification period employedby many plants), the improvement of DX by the spike was as high as0.4%-0.6%.

TABLE 4 Peak DX, Peak time and DX@48 h of AMG + Pul blend spiked withAA, compared with benchmarks Peak DX Peak Time DX@48 h [%] [hr] [%]Benchmark 95.0 48 95.0 AMG + Pul blend 96.1 72 95.7 AMG + Pul blend, AAspiked 96.3 60 96.1

CONCLUSION

The results presented above confirm that the delayed dosing ofAlpha-amylase to a blend of glucoamylase and pullulanase can furtherimprove both DX and saccharification speed.

Example 4

Maltodextrin powder from corn starch liquefaction was dissolved in waterwhile heating to make slurry starch slurry at 37.2% dry solids. Thesolid content of the slurry was measured using Refractive indexmeasurement showing 1.3982. The slurry was adjusted to a pH of 4.3 usinga 1M Hydrochloric acid solution. 18 gram aliquots of this slurry wereadded to 18 glass reaction scintillation vials with septum cap closuresand were inserted in a heating block to be heated to a temperature of61° C. Each vial was given an enzyme dosage based on the table below andadditional water was added to each vial to reach a target dry solid of36%. 1.5 mL samples were taken via needles through the septum from eachvial at different time points and were deactivated at 105° C. for 5minutes. 1 mL of each deactivated sample was diluted with 4 mL deionizedwater. The diluted samples were evaluated using a HPLC method DP1-4 formeasuring dextrose purity (% DP1 or % DX).

Table 5 shows the dextrose purity of syrup at different hours. Theresults show that addition of bacterial alpha amylase when NPUN/KNUT ismore than 60, has a positive effect on speed and % DX.

TABLE 5 Dextrose purity AGU/ NPUN/ AFAU/ KNU/ NPUN/ NPUN/ NPUN/ % DX atdifferent hour Enter sample times gDS gDS gDS gDS AGU AFAU KNU 24 hr 36hr 42 hr 48 hr 60 hr Dextrozyme DX 2X 0.18 0.36 0 0 2 N.A. N.A. 93.395.3 95.4 95.4 95.4 Dextrozyme DX 2X + 0.18 0.36 0 0.075 2 N.A. 4.8 94.195.4 95.5 95.4 95.4 Alpha amylase (SEQ ID NO: 25) AMG (SEQ ID NO: 1) +0.25 0.96 0.001 0 3.84 960 N.A. 93.3 95.6 95.8 95.8 95.8 Pullulanase(SEQ ID NO: 13) AMG (SEQ ID NO: 1) + 0.25 0.96 0.001 0.0375 3.84 96025.6 93.5 95.7 95.8 95.7 95.7 Pullulanase (SEQ ID NO: 13) + Alphaamylase (SEQ ID NO: 25) AMG (SEQ ID NO: 1) + 0.25 0.96 0.001 0.0750 3.84960 12.8 93.3 95.6 95.6 95.7 95.8 Pullulanase (SEQ ID NO: 13) + Alphaamylase (SEQ ID NO: 25) AMG (SEQ ID NO: 12) + 0.25 2.52 0 0 10.08 N.A.N.A. 93.1 95.9 96.1 96.1 96.2 Pullulanase (SEQ ID NO: 13) AMG (SEQ IDNO: 12) + 0.25 2.52 0 0.0375 10.08 N.A. 67.2 94.6 96.0 96.1 96.2 96.1Pullulanase (SEQ ID NO: 13) + Alpha amylase (SEQ ID NO: 25) AMG (SEQ IDNO: 12) + 0.25 2.52 0 0.0750 10.08 N.A. 33.6 94.5 96.0 96.0 96.1 96.1Pullulanase (SEQ ID NO: 13) + Alpha amylase (SEQ ID NO: 25)

Example 5

Saccharification by enzyme blends with delayed dosing of fungalalpha-amylases was evaluated to see how the timing of alpha-amylaseaddition affects the DX during saccharification.

Saccharification test was carried out using a Gilson Liquid handler,with which 36% DS of DE11 maltodextrin made via Liquozyme Supra was pHadjusted to 4.3 and was saccharified at 60° C. Prior to saccharificationany residual alpha-amylase activity in the maltodextrin was inactivatedby heat treatment.

Initially, a blend of glucoamylase (SEQ ID NO: 12) and pullulanase (SEQID NO: 13) containing no alpha-amylase side activity (AMG+Pul blend) wasadded to be 0.194 AGU/gDS for glucoamylase and 2.25 NPUN/gDS forpullulanase. At the same time, the DS of the maltodextrin syrup became33% by dilution caused by the enzyme addition. After different periods(0-24 h) of incubation, purified alpha-amylase (SEQ ID NO: 18) was dosedto be 15 μg/gDS corresponding to 0.01 FAU(A)/gDS, and compared with thecontrol without alpha-amylase dosing

(“No AA addition”). The sample volume increase by the alpha-amylasespike was only 0.2% so that the change in DS is negligible. After 36 hfrom the start of incubation with AMG+Pul blend, 0.75 ml aliquot of thesample was taken and the ratio of DX (glucose), DP2, DP3 and DP4+ of thesyrup was analyzed by HPLC. The timings of alpha-amylase dosing and theDX at 36 h are shown in Table 6.

TABLE 6 Timings of alpha-amylase (SEQ ID NO: 18) dosing and theresultant DX at 36 h AA dosed at DX@36 h No AA addition 94.9 0 h 95.4 2h 95.6 4 h 95.9 8 h 96.0 16 h  96.1 24 h  96.0In the same experiment batch, another purified fungal alpha-amylaseAM4279 purified from Aspergillus terreus (mature polypeptide of SEQ IDNO: 26) was spiked to be 15 μg/gDS instead of the alpha amylase setforth in SEQ ID NO: 18 at 0 h and 24 h. The timings of alpha-amylasedosing and the DX at 36 h are shown in Table 7.

TABLE 7 Timings of alpha-amylase (mature polypeptide of SEQ ID NO: 26)dosing and the re-sultant DX at 36 h. AA dosed at DX@36 h No AA addition94.9 0 h 95.2 24 h  95.4

In the same experiment batch, another AMG+Pul blend with glucoamylasefrom Aspergillus niger (mature polypeptide of SEQ ID NO: 27) instead ofthe glucoamylase set forth in SEQ ID NO: 12 was initially dosed to be0.18 AGU/gDS for glucoamylase while the pullulanase dosage was kept tobe 2.25 NPUN/gDS. The timings of alpha-amylase (SEQ ID NO: 18) dosingand the DX at 36 h are shown in Table 8.

TABLE 8 Timings of alpha-amylase (SEQ ID NO: 18) dosing and theresultant DX at 36 h AA dosed at DX@36 h No AA addition 94.9 0 h 95.9 24h  96.2

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A composition comprising a glucoamylase and a pullulanase, whereinthe ratio of pullulanase activity expressed as NPUN/g to glucoamylaseactivity expressed as AGU/g is higher than
 6. 2.-9. (canceled)
 10. Thecomposition according to claim 1, wherein the glucoamylasecomprises/consists essentially of/consists of an amino acid sequenceselected from the group consisting of i) the amino acid sequence setforth in any one of SEQ ID NO: 2-12 and 27 or a mature polypeptidethereof; ii) a subsequence of the amino acid sequence set forth in anyone of SEQ ID NOs: 2-12 and 27 or of said mature polypeptide thereof;iii) an amino acid sequence, which has at least 70% sequence identity toany one of the amino acids sequences set forth in i) and ii).
 11. Thecomposition according to claim 1, wherein the alpha-amylasecomprises/consists essentially of/consists of an amino acid sequenceselected from the group consisting of i) the amino acid sequence setforth in any one of SEQ ID NOs: 1 and 17-26 or a mature polypeptidethereof; ii) a subsequence of the amino acid sequence set forth in anyone of SEQ ID NOs: 1 and 17-26 or of said mature polypeptide thereof;iii) an amino acid sequence, which has at least 70% sequence identity toany one of the amino acids sequences set forth in i) and ii).
 12. Thecomposition according to claim 1, wherein the pullulanasecomprises/consists essentially of/consists of an amino acid sequenceselected from the group consisting of i) the amino acid sequence setforth in any one of SEQ ID NOs: 13-16 or a mature polypeptide thereof;ii) a subsequence of the amino acid sequence set forth in any one of SEQID NOs: 13-16 or of said mature polypeptide thereof; iii) an amino acidsequence, which has at least 70% sequence identity to any one of theamino acids sequences set forth in i) and ii).
 13. The compositionaccording to claim 1, said composition being a liquid or solidcomposition.
 14. A process for producing glucose syrup comprising, i)providing a composition comprising liquefied starch, and adding to saidcomposition a glucoamylase (AMG) and a pullulanase; ii) subjecting thecomposition to incubation under conditions allowing starchhydrolysis/saccharification, and subsequently iii) adding analpha-amylase to the said composition and subjecting the composition toincubation under conditions allowing starch hydrolysis/saccharification.15. The process according to claim 14, wherein the amount ofglucoamylase added in step i) corresponds to 0.05-0.5 AGU/gDS.
 16. Theprocess according to claim 14, wherein the amount of pullulanase addedin step i) corresponds to 0.05-5 NPUN(X)/gDS.
 17. The process accordingto claim 14, wherein the amount of alpha-amylase added in step iii)corresponds to 0.0005-0.025 (FAU)(A)/gDS if said alpha-amylase is afungal alpha-amylase and to 0.0005-0.025 KNU/gDS if said alpha-amylaseis a bacterial alpha-amylase. 18.-23. (canceled)
 24. The processaccording to claim 14, wherein said incubation in step ii) of claim 14has a duration of from 2-35 hours.
 25. The process according to claim14, wherein said incubation in step iii) of claim 14 has a duration offrom 2-94 hours. 26.-38. (canceled)
 39. The process according to claim14, wherein the incubation in step iii) is allowed to proceed until theamount of glucose (% DX) in said composition has reached a levelcorresponding to 95% (w/w) or more.
 40. The process according to claim14, wherein the duration of the process from addition of said aglucoamylase (AMG) and said pullulanase to the composition comprisingliquefied starch until termination of the starchhydrolysis/saccharification is from 24-96 hours.
 41. The processaccording to claim 14, wherein the starch hydrolysis/saccharificationtakes place at a pH which is within the range of 3.5-5.0, and at atemperature, which is within the range of 59-70° C.
 42. The processaccording to claim 14, wherein the composition comprising liquefiedstarch provided in step i) contains from 25-45% dry solids (% DS). 43.(canceled)
 44. The process according to claim 14, wherein theglucoamylase comprises/consists essentially of/consists of an amino acidsequence selected from the group consisting of i) the amino acidsequence set forth in any of SEQ ID NO: 2-12 and 27 or a maturepolypeptide thereof; ii) a subsequence of the amino acid sequence setforth in any one of SEQ ID NOs: 2-12 and 27 or of said maturepolypeptide thereof; iii) an amino acid sequence, which has at least 70%sequence identity to any one of the amino acids sequences set forth ini) and ii).
 45. The process according to claim 14, wherein thealpha-amylase comprises/consists essentially of/consists of an aminoacid sequence selected from the group consisting of i) the amino acidsequence set forth in any of SEQ ID NOs: 1 and 17-26 or a maturepolypeptide thereof; ii) a subsequence of the amino acid sequence setforth in any one of SEQ ID NOs: 1 and 17-26 or of said maturepolypeptide thereof; iii) an amino acid sequence, which has at least 70%sequence identity to any one of the amino acids sequences set forth ini) and ii).
 46. The process according to claim 14, wherein thepullulanase comprises/consists essentially of/consists of an amino acidsequence selected from the group consisting of i) the amino acidsequence set forth in any one of SEQ ID NOs: 13-16 or a maturepolypeptide thereof; ii) a subsequence of the amino acid sequence setforth in any one of SEQ ID NOs: 13-16 or of said mature polypeptidethereof; iii) an amino acid sequence, which has at least 70% sequenceidentity to any one of the amino acids sequences set forth in i) andii).
 47. A process for producing glucose syrup from liquefied starch,comprising contacting the liquefied starch with a composition accordingto claim
 1. 48. The process according to claim 47, wherein saidliquefied starch contains from 25-45% dry solids (% DS), such as from25-40% DS from 30-38% DS, from 32-38% DS, or such as from 34-36% DS.